Antiangiogenic small molecules and methods of use

ABSTRACT

Methods of inhibiting undesired angiogenesis are provided, which methods include administering to a subject a therapeutically effective amount of at least one of the compounds described herein, or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATION

This is the U.S. National Stage of International Application No.PCT/US2010/043998, filed Jul. 30, 2010, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/230,667, filed Jul. 31, 2009, which isincorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to antiangiogenic compounds, derivativesthereof, and methods of use of such compounds and derivatives.

BACKGROUND

Angiogenesis is the process of formation of new blood vessels fromexisting ones. Angiogenesis is a normal and vital process in growth anddevelopment, as well as in pathological conditions. Angiogenesis hasbeen intensively studied over the past several decades because of itsfundamental importance in tissue development, vascular diseases, andcancer. Under normal physiological conditions, humans or animals undergoangiogenesis only in very specific restricted situations. For example,angiogenesis is normally observed in fetal and embryonal development andformation of the corpus luteum. Post-natal angiogenesis is an importantphysiological function in the ovary, endometrium, placenta, and in woundhealing. Deregulation of angiogenesis plays a major role in many humandiseases including diabetic retinopathy, age-related maculardegeneration, endometriosis, atherogenesis, arthritis, psoriasis,corneal neovascularization, rheumatoid arthritis, tumorigenesis, andmetastasis, among others.

Tumor angiogenesis involves the proliferation of a network of bloodvessels that penetrates into cancerous growths, supplying nutrients andoxygen and removing waste products. Angiogenesis is also an element ofmetastasis of a tumor. Single cancer cells can break away from anestablished solid tumor, enter the blood vessel, and be carried to adistant site, where they can implant and begin the growth of a secondarytumor. It has even been suggested that the blood vessels in a solidtumor may in fact be mosaic vessels, comprised of both endothelial cellsand tumor cells. Such mosaicity allows for substantial shedding of tumorcells into the vasculature.

It has been shown that there is a direct correlation between tumormicrovessel density and the incidence of metastasis. Tumor cellsthemselves can produce factors that stimulate the proliferation ofendothelial cells and new capillary growth. Angiogenesis is important intwo stages of tumor metastasis: in vascularization of the tumor, whichallows tumor cells to enter the blood stream and to circulate throughoutthe body; and after the tumor cells have left the primary site andsettled into the secondary (metastasis) site, angiogenesis must occurbefore the new tumor can grow and expand. Therefore, prevention ofangiogenesis could lead to the prevention of metastasis of tumors andpossibly contain the neoplastic growth at the primary site.

Blockage of angiogenesis is recognized as one of the most promisingstrategies against cancer (including metastases), retinopathy andendometriosis, among other diseases. A significant increase in theresearch effort in the angiogenesis field over the past decade hasresulted in a substantial increased understanding of the angiogenicprocess and subsequently the development of new therapeutics to modulateangiogenesis. Because of their extended biological half-life, highdiffusibility coefficient and cost effective synthesis non-peptidicantiangiogenic small molecules (SMs) are the main focus ofpharmaceutical companies and academic institutions.

Angiogenesis-based anti-tumor therapies typically use natural andsynthetic angiogenesis inhibitors such as angiostatin, endostatin andtumstatin. Recently the Food and Drug Administration (FDA) approved anantibody therapy targeting angiogenesis in colorectal cancer. Thistherapy is based on a monoclonal antibody directed against an isoform ofVEGF and is marketed under the trade name Avastin®. The pharmaceuticalindustry has focused in the development tyrosine kinase inhibitors andtubulin binders as antiangiogenic small molecules. Thus, a need existsfor small molecules which exploit other angiogenesis pathways.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to the identification of a new set ofantiangiogenic small molecule inhibitors. Cell-based high throughputscreenings (HTS) together with chemo-informatic tools were applied tothe discovery of antiangiogenic small molecules. Rather than targetingthe HTS to a single subcellular molecule, the screen described hereintargeted the entire cellular process of angiogenesis. In particular, twocell based assays were employed, which represent the two most importantsteps in angiogenesis: endothelial cell growth and tube formation.

As a result, a new set of antiangiogenic small molecules (SMs) have beendiscovered. Structure-activity-relationship (SAR) studies have shownthat the majority of the newly identified bioactive SMs are not relatedto previously recognized antiangiogenic SMs, based on comparisons tovarious databases (e.g., FDA marketed compounds; SMs currently inclinical trials compounds; and SMs annotated as antiangiogenic inchemical databases compounds, PubChem, LeadScope, DrugBank, DTP/NCI,etc.).

Based on the identification of this new set of antiangiogenic SMs,disclosed herein are methods for inhibiting angiogenesis (particularlyundesired angiogenesis) in a subject that include administering to asubject a therapeutically effective amount of at least oneantiangiogenic compound (e.g., antiangiogenic small molecule) from amongthe compounds referred to herein as Compounds 1-77, or pharmaceuticallyacceptable salts thereof, examples of which are described in detailbelow.

Also disclosed herein are methods of inhibiting angiogenesis thatinclude administering to a subject a therapeutically effective amount ofat least one of the compounds referred to herein as Compounds 1-77, orpharmaceutically acceptable salts thereof, examples of which aredescribed in detail below.

Further disclosed herein are compounds having a structure represented byany one of the formulas shown in TABLE 1 (referred to herein asCompounds 1-77), and pharmaceutically acceptable salts thereof.Pharmaceutical compositions that include the above-described compoundsare also disclosed herein.

Thus, disclosed herein are pharmaceutical compositions for treating anangiogenesis-dependent disease, comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically-acceptable salt thereof. In particular examples, thepharmaceutical composition further comprises[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof.

Also disclosed herein are pharmaceutical compositions for inhibitingaberrant angiogenesis or inhibiting growth of neoplasitic tissuecomprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically-acceptable salt thereof. In particular examples, thepharmaceutical composition further comprises[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof.

Also disclosed herein are methods of treating an angiogenesis-dependentdisease, comprising: administering to a subject having or predisposed toan angiogenesis-dependent disease a therapeutically effective amount ofa composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. In particular embodiments ofthe methods, the composition further comprises[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof.

Further disclosed herein are methods of inhibiting undesiredangiogenesis in a subject, comprising: identifying a subject whereinangiogenesis is not desired, and administering to the subject atherapeutically effective amount of a composition comprising at leastone of 2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC150117), deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methylpropanoate (NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC122657), maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. In particular examples of thedisclosed methods, the composition further comprises[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof.

Also disclosed are methods of inhibiting a neoplasm in a subject,comprising: administering to the subject a therapeutically effectiveamount of a composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. In particular examples, thecomposition further comprises [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof.

Further disclosed are methods of inhibiting angiogenesis in a tissue ora target area in a subject wherein the formation of new blood vessels isnot desired, comprising identifying a tissue or target area in a subjectwherein the formation of new blood vessels is not desired; andintroducing directly or indirectly into the tissue or target area aneffective amount of a composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof, thereby inhibitingangiogenesis in the tissue or target area. In particular examples, thecomposition further comprises [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof.

The foregoing and other objects, features, and advantages will becomemore apparent from the following detailed description, which proceedswith reference to the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a summarized workflow utilized for the identification ofthe new set of antiangiogenic SMs described herein. Additional detailsare provided in Example 1.

FIG. 2 shows the basic experimental design for the growth and tubeformation HTS assays.

FIG. 3 shows the results on one of the plates (4143-11) included in thegrowth HTS using porcine aortic endothelial (PAE) cells. The plotrepresents fluorescence emission of all 96 wells over 7 days; asexpected, fluorescence values increase over time although differentwells show different fluorescence values. A composite image of all 96wells is also shown (at day 4 of growth). Positive controls are shown inthe far right column of wells; as expected, these controls show maximumvalues of fluorescence (in the plot) and high density of cells in thewells. A negative control is shown in the left column of wells; theseshow low fluorescence values in the plot and low cell density in thewells. Additionally, an example of a compound which blocks growth of PAEis shown in the first row, fourth column from the left, with lowfluorescence values and few cells in the well. Furthermore, an exampleof a compound which does not inhibit the growth of PAE is shown in theseventh row, fourth column. In this case, high fluorescence values areshown in the plot together with a high number of cells in the well.

FIG. 4: Following the same protocol, HTS growth experiments wereperformed for PAE, BEC, A549 and MCF7 cells. These experiments weredesigned to explore the specificity of SMs with inhibitory activity inendothelial cells as compared to tumor cells from different anatomicalorigins.

The image in FIG. 4 shows a heatmap which summarizes the resultsobtained in the HTS experiments. The heatmap was constructed using thefunction heatmap.2 of the package gplots of the R statistical software.Clustering was performed using Euclidean distance matrix. The X axisshows the 1974 SMs tested and the Y axis represents some of the growthHTS experiments performed. For all experiments, measurements obtained indifferent days have been included and as expected show a high degree ofconsistency. Dark cells in the heatmap represent SMs with strongestgrowth inhibitory activity, and lighter greys are SM with no activity ongrowth.

FIG. 5: In order to study SMs which preferentially inhibit the growth ofendothelial cells or tumor cells, average growth activity values forendothelial cells and tumor cells were compared in a bivariatescatterplot. Most of the SMs do not have an effect on growth in any ofthe cell lines tested and cluster in the center of the scatterplot(indicated within the middle-sized oval). Also, most SMs with growthinhibitory activity show similar potency against tumor cells andendothelial cells and therefore cluster in the lower left quadrant(indicated within the largest oval). Interestingly, a few SMs showedgrowth inhibitory activity in tumor cells but not in endothelial cells(small oval; see also TABLE 10). Growth activity of these smallmolecules is shown in the adjacent plot (small molecules are identifiedby their position in the plate; TABLE 10 correlates plate position toNSC number).

FIG. 6: Dose response curves were constructed for all the SMs ofinterest using PAE cells. Data were fitted to non-linear sigmoid curvesusing GraphPad Prism (GraphPad Software, Inc., La Jolla, Calif.). Sincethe initial screening was performed using a final SM concentration of 1μM, IC50 for most compounds were confirmed to be in the range of 10⁻¹²to 10⁻⁹ M. This confirms that all the SMs discovered in this project arehighly potent inhibitors of endothelial cell growth.

FIG. 7: In order to explore the cytotoxic potential of the SMs ofinterest, a novel high throughput cytotoxicity assay was developed, andis further described in U.S. application Ser. No. 12/060,752 (publishedas US 2009/0088341 on Apr. 2, 2009; incorporated herein by reference inits entirety). In FIG. 7, two different hypothetical situations aregraphically described: In the lower area of the diagram, fluorescentcells are exposed to a cytotoxic substance, which results in the releaseof florescence to the cell culture medium. Both the fluorescence in themedium and the remaining fluorescence in the cells can be quantitatedand used to calculate the percentage of cytotoxicity using the formulain the lower area of the figure. The plot shows the expected doseresponse curve when Triton X is used as a cytotoxic agent on PAE cells.

FIG. 8 shows an example of four compounds with growth inhibitoryactivity, two of which present a strong and moderate cytotoxic activityrespectively. Using the cytotoxicity assay illustrated in FIG. 7 anddescribed herein, four compounds were identified as cytotoxic (NSC88903, NSC 310551, NSC 18877, and NSC 321206; see TABLE 10).

FIG. 9 shows a screen shot from the an image analysis program namedAngioApplication™, which was developed for the HTS format and permitteda morphological quantitative analysis of tube formation (described indetail in U.S. application Ser. No. 12/060,752, which published as US2009/0088341 on Apr. 2, 2009; incorporated herein by reference in itsentirety). This software is able to rapidly assess a variety of metricsin images of tube formation including (but not limited to) tube length,node area, branching points, fractal dimension and lacunarity.

FIG. 10 shows how principal component analysis was used to investigatewhich metrics explained better the variability of the data sets. Theresults showed that emptiness and branching index were the mostappropriate metrics.

FIG. 11: Emptiness (C1) and branching index (C2) were plotted in abivariate scatter plot for every SM tested. The Euclidean distancebetween the average of the positive controls and every SM was used asmetric to define anti-tube formation activity (calculations were doneseparately for every plate). In essence, compounds which are furtheraway from the positive controls are more likely to be antiangiogenic.This figure illustrates the results from all the SMs in one plate. Asexpected, most of the compounds (small squares clustered in center ofgraph) are located closely to the positive controls (large squaresclustered near center of graph). In contrast, the negative controls(large squares clustered in lower right corner of graph) are positionedfarther away from the positive controls. Representative images of thepositive and negative control cells are shown. Tube formation inhibitorycompounds are detected as being located at an intermediate distancebetween the positive controls and the negative controls. Arepresentative image of an active SM is shown. 35 out the 1974 compoundsin the library (1.75%) were found to statistically significantly inhibittube formation (TABLE 10).

FIG. 12: IC50 were calculated for all tube formation inhibitors. Asexpected, most IC50 were in the range of 10⁻⁹ to 10⁻¹²M, making thesecompounds highly effective tube formation inhibitors. This figureillustrates an example of the dose response generated with compoundNSC119889.

FIG. 13 summarizes the results obtained in the growth and tube formationHTS for endothelial cells; information related to specific compounds issummarized in TABLE 10. 2.4% (48) of the compounds were growthinhibitors, and 1.75% (35) were tube formation inhibitors. 0.5% (11) ofthe compounds showed both growth and tube formation inhibitory activity.

FIG. 14: The antiangiogenic SMs identified in this project werestructurally compared with annotated compounds in available annotated SMdatabases, such as PubChem, DrugBank, LeadScope and FDA Marketed Drugsamong others. Structural classifications were performed with LeadScopesoftware. Only a few of the SMs identified were structurally related toannotated compounds in other databases (numbers in parenthesis in theabove figure). This can be explained by the novel drug discoverymethodology utilized in this project, which, as expected, results incompounds with novel SARs. None of the antiangiogenic SMs discovered inthis project are structurally related to any known antiangiogenic SMs.This supports the novelty of the herein described antiangiogenic SMs andemphasizes that new SARs will result in exploitation of new cellularantiangiogenic pathways.

FIG. 15 is a series of graphs showing the inhibitory effects of selectedSMs on growth of tumor xenografts. Top panels show the effect of SMs ongrowth of A549 tumors. Bottom panels show the effect of SMs on growth ofSK-ML-1 tumors.

FIG. 16 is a series of graphs showing the effects of selected SMs on theinhibition of tubulin polymerization.

FIG. 17 is a series of volcano plots showing quantitative real timeRT-PCR analysis of the effects of selected SMs on expression of genesimplicated in angiogenesis

SEQUENCE LISTING

The nucleic and/or amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three letter code for amino acids, as defined in37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown,but the complementary strand is understood as included by any referenceto the displayed strand. The SEQ ID designations in the accompanyingsequence listing are set forth in Table 13. Sequences and informationassociated with the accession numbers listed on Table 14 (and availableonline at ncbi.nlm.nih.gov/guide/) are incorporated by reference hereinin their entirety.

The Sequence Listing is submitted as an ASCII text file, Annex C/St.25text file, created on Jul. 27, 2010, 68.1 KB, which is incorporated byreference herein.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Abbreviations

-   bFGF: basic fibroblast growth factor-   EC50: The term half maximal effective concentration-   FDA: Food and Drug Administration-   GFP: green fluorescent protein-   HTS: high throughput screen-   PAE: porcine aortic endothelial-   RTK: receptor tyrosine kinase-   SAR: structure-activity relationship-   SMs: small molecules-   RFP: red fluorescent protein-   VEGF: vascular endothelial growth factor-   YFP: yellow fluorescent protein

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. The term“comprises” means “includes.” The abbreviation, “e.g.” is derived fromthe Latin exempli gratia, and is used herein to indicate a non-limitingexample. Thus, the abbreviation “e.g.” is synonymous with the term “forexample.” All chemical compounds include both the (+) and (−)stereoisomers (as well as either the (+) or (−) stereoisomer), and anytautomers thereof. It is further to be understood that all molecularweight or molecular mass values given for compounds are approximate, andare provided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of this disclosure, suitable methods and materials are describedherein. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the disclosure, thefollowing explanations of specific terms are provided:

Acid: A compound capable of transferring a hydrogen atom in solution.Acid is inclusive of, but not limited to, a carboxylic acid.

Administer/Administration: To give or apply, for instance to a subject.To “introduce” is understood to be equivalent to “administer.” This termincludes topical, parenteral, oral, intravenous, intra-muscular,sub-cutaneous, inhalational, nasal, or intra-articular routes ofadministration, among others. By way of example, a therapeutic compound,such as an antiangiogenic agent, can be administered. Administration canbe local or systemic, direct or indirect.

Non-limiting examples of local administration include, but are notlimited to, topical administration, subcutaneous administration,intramuscular administration, intrathecal administration,intrapericardial administration, intra-ocular administration, topicalophthalmic administration, or administration to the nasal mucosa orlungs by inhalational administration. In addition, local administrationincludes routes of administration typically used for systemicadministration, for example by directing intravascular administration tothe arterial supply for a particular organ or tumor. Thus, in particularembodiments, local administration includes intra-arterial administrationand intravenous administration when such administration is targeted tothe vasculature supplying a particular organ or tumor.

Systemic administration includes any route of administration designed todistribute the administered compound widely throughout the body via thecirculatory system. Thus, systemic administration includes, but is notlimited to, intra-arterial and intravenous administration. Systemicadministration also includes, but is not limited to, topicaladministration, subcutaneous administration, intramuscularadministration, or administration by inhalation, when suchadministration is directed at absorption and distribution throughout thebody by the circulatory system.

Direct administration or introduction involves the direct contact of acompound to a target are, such as by injection. Indirect administrationor introduction involves any other method other than direct contact ofthe compound, for example by oral ingestion.

Alkyl: A branched or straight chain alkyl group containing only carbonand hydrogen. In certain embodiments, alkyl groups may contain one totwelve carbon atoms, particularly one to six carbon atoms. This term isfurther exemplified by groups such as methyl, ethyl, n-propyl, isobutyl,t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkylgroups can either be unsubstituted or substituted with one or moresubstituents, e.g., halogen, alkoxy, cycloalkyl, alkylthio,trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl,arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino,piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

Amino acid moiety: A moiety that contain one or more primary, secondaryor tertiary amino groups and one or more acidic carboxyl groups (—COOH)or a moiety that is a derivative or residue of an amino acid in thesense that the moiety contains one or more amino groups (e.g., —NH₂) andone or more ester groups (i.e., —OC(O)—).

Animal: A living multi-cellular vertebrate organism, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, pigs, rats, mice, and cows.

Angiogenesis: A biological process leading to the generation of newblood vessels through sprouting and/or growth from pre-existing bloodvessels. The process can involve the migration and proliferation ofendothelial cells from preexisting vessels. Angiogenesis occurs duringpre-natal development, post-natal development, and in the adult. In theadult, angiogenesis occurs during the normal cycle of the femalereproductive system, wound healing, and during pathological processessuch as cancer (for a review see Battegay, J. Molec. Med. 73(7):333-346, 1995).

Angiogenic activity: The ability of an agent to promote or inhibitangiogenesis. Angiogenic activity can be measured in an angiogenesisassay, for example using the fluorescent cell-lines and assays disclosedherein and/or in U.S. application Ser. No. 12/060,752 (published as US2009/0088341 on Apr. 2, 2009; and incorporated herein by reference inits entirety).

Angiogesis-dependent disease: A disease that is at least partiallydependent on the stimulation of aberrant (undesired) angiogenesis forits progression. Aberrant angiogenesis can result from misexpression ofangiogenic factors in otherwise normal cells. Aberrant angiogenesis canalso be stimulated by tumors producing one or more angiogenic factors.

Angiogenic factor: A molecule that affects angiogenesis, for example bystimulating or inhibiting angiogenesis. Numerous experiments havesuggested that tissues secrete factors that promote angiogenesis underconditions of poor blood supply during normal and pathologicalangiogenesis processes. The formation of blood vessels is initiated andmaintained by a variety of factors secreted either by a cell (such as atumor cell) or by accessory cells. Many different growth factors andcytokines have been shown to exert chemotactic, mitogenic, modulatory orinhibitory activities on endothelial cells, smooth muscle cells andfibroblasts and can, therefore, be expected to participate in anangiogenic process. For example, factors modulating growth, chemotacticbehavior and/or functional activities of vascular endothelial cellsinclude aFGF, bFGF, angiogenin, angiotropin, epithelial growth factor,IL-8, and vascular endothelial growth factor (VEGF) among others.

Because many angiogenic factors are mitogenic and chemotactic forendothelial cells, their biological activities (such as angiogenicactivities) can be determined in vitro by measuring the inducedmigration of endothelial cells or the effect of these factors onendothelial cell proliferation using the cell-lines assays and methodsdisclosed herein. For example, migration assays and other assays, suchas tubule formation assays and growth assays can also be used todetermine angiogenic activity, for example the angiogenic activity inthe presence of a test agent, such as a potential angiogenesisinhibitor.

Aryl: A monovalent unsaturated aromatic carbocyclic group having asingle ring (e.g., phenyl) or multiple condensed rings (e.g., naphthylor anthryl), which can optionally be unsubstituted or substituted with,e.g., halogen, alkyl, alkoxy, mercapto (—SH), alkylthio,trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, anotheraryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino,morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or otherfunctionality.

Biological sample: A sample obtained from a plant or animal subjectabout which information is desired, for example, information about thesamples ability to promote cellular growth, tubule formation, and/orcellular migration. As used herein, biological samples include allclinical samples, including, but not limited to, cells, tissues, andbodily fluids, such as: blood; derivatives and fractions of blood, suchas serum, and lymphocytes (such as B cells, T cell, and subfractionsthereof); extracted galls; biopsied or surgically removed tissue,including tissues that are, for example, unfixed, frozen, fixed informalin and/or embedded in paraffin; tears; milk; skin scrapes; surfacewashings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; bonemarrow aspirates; middle ear fluids, bronchoalveolar levage, trachealaspirates, sputum, nasopharyngeal aspirates, oropharyngeal aspirates, orsaliva. In particular embodiments, the biological sample is obtainedfrom an animal subject, such as in the form of middle ear fluids,bronchoalveolar levage, tracheal aspirates, sputum, nasopharyngealaspirates, oropharyngeal aspirates, or saliva. In particularembodiments, the biological sample is obtained from a subject, such asblood or serum. A patient sample is a sample obtained from a subject,such as a mammalian subject, for example a human subject under medicalcare.

Cellular activity: An activity of a particular cell-line, such as theability of the cell to divide, migrate in response to stimulus, or toform three dimensional structures, such as tubules. Cellular activity(s)of a particular cell-line can be assessed using in vitro assays, forexample the assays disclosed herein.

Cancer: A malignant disease characterized by the abnormal growth anddifferentiation of cells. “Metastatic disease” refers to cancer cellsthat have left the original tumor site and migrate to other parts of thebody for example via the bloodstream or lymph system.

Examples of hematological tumors include leukemias, including acuteleukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia, andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (such as adenocarcinoma), lung cancers,gynecological cancers (such as, cancers of the uterus (e.g., endometrialcarcinoma), cervix (e.g., cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (e.g., ovarian carcinoma, serous cystadenocarcinoma,mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clearcell carcinoma, unclassified carcinoma, granulosa-thecal cell tumors,Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva(e.g., squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (e.g., clear cellcarcinoma, squamous cell carcinoma, botryoid sarcoma), embryonalrhabdomyosarcoma, and fallopian tubules (e.g., carcinoma)), prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma), and skin cancer (such as melanoma and non-melanoma).

Cell culture: The process by which either prokaryotic or eukaryoticcells are grown under controlled conditions. In practice the term “cellculture” has come to refer to the culturing of cells derived frommulticellular eukaryotes, especially animal cells, such as mammaliancells, for example the fluorescent cells disclosed herein. Mammaliancells are grown and maintained at an appropriate temperature and gasmixture (typically, 37° C., 5% CO₂) in a cell incubator. Cultureconditions vary widely for each cell type, and variation of conditionsfor a particular cell type can result in different phenotypes beingexpressed. Aside from temperature and gas mixture, the most commonlyvaried factor in culture systems is the growth medium. Recipes forgrowth media can vary in pH, glucose concentration, growth factors, andthe presence of other nutrient components. The growth factors used tosupplement media are often derived from animal blood, such as calfserum.

Some cells naturally live without attaching to a surface, such as cellsthat exist in the bloodstream. Others require a surface, such as mostcells derived from solid tissues. Cells grown unattached to a surfaceare referred to as suspension cultures. Other adherent cultures cellscan be grown on tissue culture plastic, which may be coated withextracellular matrix components (for example collagen or fibronectin) toincrease its adhesion properties and provide other signals needed forgrowth. “Co-culture” refers to the culture of more than one cell-line(such as more than one of the disclosed cell-lines) in a single vessel.Co-cultures can be 2-dimensional (2-D) or 3-dimensional (3-D). Examplesof both 2-D and 3-D co-cultures are described in U.S. patent applicationSer. No. 12/802,666, filed on Jun. 10, 2010 *published as now U.S. Pat.No. 8,679,836 on Mar. 25, 2014).

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an angiogenesis inhibitor.Chemotherapeutic agents are described for example in Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc;Baltzer and Berkery. (eds): Oncology Pocket Guide to Chemotherapy, 2nded. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds):The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book,1993. Combination chemotherapy is the administration of more than oneagent to treat cancer, for example an alkylating agent and anangiogenesis inhibitor.

Contacting: To place in direct physical association, including in solidor in liquid form. Contacting can occur in vivo, for example byadministering an agent to a subject, or in vitro for example withisolated cells or cell-cultures, for example cell-cultures of thedisclosed fluorescent cell-lines.

Control: A reference standard. A control can be a known value indicativeof basal cellular activity, such as basal migratory potential, doublingtime, tubule formation potential and the like, or a controlcell-culture, such as a culture including at least one of the disclosedfluorescent cell-lines, not treated with an exogenous agent, such as atest agent, one or more cell-lines (such as the fluorescent cell-linesdisclosed herein), angiogenic factor, angiogenic inhibitor, or the like.A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 10%, such as at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 100%, at least about 150%, at least about 200%, atleast about 250%, at least about 300%, at least about 350%, at leastabout 400%, at least about 500%, or greater then 500%.

Cycloalkyl: Includes a moiety that contains at least one cycloalkyl ringstructure. There may be one or more ring structures including a bridgedcyclic structure or a fused ring structure. The cycloalkyl may beunsubstituted or substituted with one or more substituents, e.g.,halogen, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto,carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino,dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl,or other functionality. Illustrative cycloalkyl groups includecyclopropyl, cyclopentyl, cyclohexyl, cyclobutyl, and decahydronaphthyl.

EC50: The term half maximal effective concentration (EC50 or EC₅₀)refers to the concentration of a drug which induces a response halfwaybetween the baseline and maximum. EC50 is commonly used as a measure ofdrug potency.

Electromagnetic radiation: A series of electromagnetic waves that arepropagated by simultaneous periodic variations of electric and magneticfield intensity, and that includes radio waves, infrared, visible light,ultraviolet light, X-rays and gamma rays. In particular examples,electromagnetic radiation is emitted by a laser, which can possessproperties of monochromaticity, directionality, coherence, polarization,and intensity. Lasers are capable of emitting light at a particularwavelength (or across a relatively narrow range of wavelengths), forexample such that energy from the laser can excite one fluorophore witha specific excitation wavelength but not excite a second fluorophorewith a specific excitation wavelength difference and distinct from theexcitation wavelength on the first fluorophore.

Emission or emission signal: The light of a particular wavelengthgenerated from a source. In particular examples, an emission signal isemitted from a fluorophore, such as a fluorescent protein, after thefluorophore absorbs light at its excitation wavelength(s).

Excitation or excitation signal: The light of a particular wavelengthnecessary and/or sufficient to excite an electron transition to a higherenergy level. In particular examples, an excitation is the light of aparticular wavelength necessary and/or sufficient to excite afluorophore, such as a fluorescent protein, to a state such that thefluorophore will emit a different (such as a longer) wavelength of lightthen the wavelength of light from the excitation signal.

Exogenous agent: An exogenous agent is any agent external to a targetcell-line that is to be studied, and it includes small molecules,proteins, biological samples (such as patient samples) and other cellsor cell-lines, such as fluorescent cell-lines other than the targetcell-line, for example a different type of cell that can by identifiedas different by a distinguishable fluorescent signal.

Expression: With respect to a gene sequence, refers to transcription ofthe gene and, as appropriate, translation of the resulting mRNAtranscript to a protein. Thus, expression of a protein coding sequence,such as the expression of a fluorescent protein, results fromtranscription and translation of the coding sequence for that protein.Constitutive expression refers to the expression of a gene product, suchas a protein, for example a fluorescent protein, in a substantialcontinuous manner, such that the expression is not interrupted. Anexample of constitutive expression is continuous expression in theabsence of an exogenous stimulating agent, such as an agent used toactivate a promoter. Stable expression refers to expression that is notlost or reduced substantially over time, for example expression thatdoes not diminish through multiple passages of a cell-line, for examplea cell-line constitutively expressing a fluorescent protein.

Fluorescent property: A characteristic of a fluorescent molecule, suchas a fluorescent protein, for example green fluorescent protein, redfluorescent protein, yellow fluorescent protein, cyan fluorescentprotein and the like. Examples of fluorescent properties include themolar extinction coefficient at an appropriate excitation wavelength,the fluorescence quantum efficiency, the shape of the excitationspectrum or emission spectrum (the “fluorescence spectrum,” theexcitation wavelength maximum and emission wavelength maximum, the ratioof excitation amplitudes at two different wavelengths, the ratio ofemission amplitudes at two different wavelengths, the excited statelifetime, or the fluorescence anisotropy. Quantifying fluorescencerefers to the determination of the amount of fluorescence generated by afluorophore, for example a fluorescent protein, which can be thequantity of photons emitted by a fluorophore. In some examples,fluorescence is quantified by measuring the intensity of a fluorescencesignal at a particular wavelength, for example the wavelength of theemission maxima of a particular fluorophore, such as a fluorescentprotein. Fluorescence intensity can also be quantified at a wavelengththat is not the emission maxima of a particular fluorophore, for exampleto avoid emission spectra that overlap and thereby interfere with theemission maxima of a particular fluorophore, such as a particularfluorescent protein. In some examples, a fluorescence signal is emittedby a population of fluorescent proteins, for example fluorescentproteins present in a population of cells containing such fluorescentproteins. Such a signal can be quantified, for example to determine thenumber, or relative number of cells that emit such a fluorescent signal.Detecting a pattern of fluorescence refers to the correlation of afluorescent signal to a specific location to determine the locationwhere a fluorescence signal, such as a fluorescent signal of aparticular wavelength, originates. In some examples, a pattern offluorescence determines the location and or shape of the cells that emita fluorescence signal, such as cells containing a fluorescent protein,for example to determine the number of the total area of the tubules,the total number of tubules, number of nodes, number of branch points,the number of tubes per node, or node area formed by such cells usingthe methods disclosed herein, and disclosed in U.S. application Ser. No.12/060,752 (published as US 2009/0088341 on Apr. 2, 2009; andincorporated herein by reference in its entirety).

Fluorescent protein: A protein capable of emission of a detectablefluorescent signal. Fluorescent proteins can be characterized by thewavelength of their emission spectrum. For example green fluorescentprotein (GFP) has a fluorescent emission spectrum in the green part ofthe visible spectrum. In addition to green-fluorescent proteins,fluorescent proteins are known which fluoresce in other regions of thevisible spectrum, for example blue-fluorescent proteins,cyan-fluorescent proteins, yellow-fluorescent proteins,orange-fluorescent proteins, red-fluorescent proteins, and far-redfluorescent proteins. Examples of fluorescent proteins can be found inthe following patent documents: U.S. Pat. Nos. 5,804,387; 6,090,919;6,096,865; 6,054,321; 5,625,048; 5,874,304; 5,777,079; 5,968,750;6,020,192; 6,146,826; 6,969,597; 7,150,979; 7,157,565; and 7,166,444;and published international patent applications WO 07/085,923; WO07/052,102, WO 04/058973, WO 04/044203, WO 03/062270; and WO 99/64592.Additional examples of fluorescent proteins are available fromClonetech, Laboratories, Inc. (Mountain View, Calif.) under the tradename Living Colors®. Nucleic acids encoding such fluorescent proteinscan be incorporated into mammalian expression vectors for use inproducing the disclosed fluorescent cell-lines.

Growth rate: The expansion of the number of cells of a specifiedcell-line through cell division as a function of time. In one examplethe growth rate is the rate at which a cell-line grown in culturedoubles.

Halogen: Refers to fluoro, bromo, chloro and iodo substituents.

High throughput technique: Through this process one can rapidly identifyactive compounds, antibodies or genes which affect a particularbiomolecular pathway, for example pathways in angiogenesis. In certainexamples, combining modern robotics, data processing and controlsoftware, liquid handling devices, and sensitive detectors, highthroughput techniques allows the rapid screening of potentialpharmaceutical agents in a short period of time.

Histology: The study of the microscopic anatomy and classification oftissue, including the histology of mammalian cells, such as cells andcell-lines from mammalian tissues. Histological typing refers to thecategorizing of tissue into histological types, for example bymicroanatomical origin (such as connective tissue, nerves, muscles, andcirculatory cells, among others) or cell-types (such as epithelialcells, stromal cells among others). Cells can be classified as being ofdifferent histological types by virtue of the staining and/or reactionwith antibodies, or by characteristic microanatomical features. Cells ofdifferent histological types interact differently with different stainsand/or antibodies. Methods for histological typing are well known in theart. Histology can be use to determine if cells are of different types.Thus, in some examples different cell-lines are histologically differentcell-lines.

Immortalized cell or cell-line: A cell or cell-line that has acquiredthe ability to proliferate indefinitely either through random mutationor deliberate modification, such as artificial expression of thetelomerase gene. There are numerous well established immortalizedcell-lines representative of particular cell types.

Inhibitor (for example, of angiogenesis): A substance capable ofinhibiting [something] to some measurable extent, for exampleangiogenesis. In disclosed examples, inhibition of angiogenesis ismeasured in one of the assays disclosed herein.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects, forexample, humans, non-human primates, mice, rats, dogs, cats, horses, andcows.

Migration potential: The ability of cells, such as the cell-linedisclosed herein, to translocate in response to a chemical stimulus,such as a growth factor. Migration potential can be determined with theassays disclosed herein.

Mixed cell population: A population of cells, such as cells in culture,that contains two or more different types of cells, such ashistologically different cell-lines. Examples of different types ofcells include cells of different embryonic origin (such as cellsoriginating from the ectoderm, endoderm, or mesoderm), cells fromdifferent cellular locations (such as cells from epithelium,endothelium, or stroma), cells from different tissues or organs (such ascells from pulmonary myocardial, neural, vascular, skin, bone, orskeletal or smooth muscle tissue).

Neoplasm or tumor: Any new and abnormal growth; particularly a newgrowth of tissue in which the growth is uncontrolled and progressive. Aneoplasm, or tumor, serves no useful function and grows at the expenseof the healthy organism.

In general, tumors appear to be caused by abnormal regulation of cellgrowth. Typically, the growth of cells in the body is strictlycontrolled; new cells are created to replace older ones or to performnew functions. If the balance of cell growth and death is disturbed, atumor may form. Abnormalities of the immune system, which usuallydetects and blocks aberrant growth, also can lead to tumors. Othercauses include radiation, genetic abnormalities, certain viruses,sunlight, tobacco, benzene, certain poisonous mushrooms, and aflatoxins.

Tumors are classified as either benign (slow-growing and usuallyharmless depending on the location), malignant (fast-growing and likelyto spread and damage other organs or systems) or intermediate (a mixtureof benign and malignant cells). Some tumors are more common in men orwomen, some are more common amongst children or elderly people, and somevary with diet, environment and genetic risk factors.

Symptoms of neoplasms depend on the type and location of the tumor. Forexample, lung tumors can cause coughing, shortness of breath, or chestpain, while tumors of the colon can cause weight loss, diarrhea,constipation and blood in the stool. Some tumors produce no symptoms,but symptoms that often accompany tumors include fevers, chills, nightsweats, weight loss, loss of appetite, fatigue, and malaise.

Blood vessels supply tumors with nutrients and oxygen. Tumor growth isdependent on the generation of new blood vessels that can maintain theneeds of the growing tumor, and many tumors secrete substances(angiogenic factors) that are able to induce proliferation of new bloodvessels (angiogenesis). Anti-tumor therapies include the use ofangiogenesis inhibitors, which reduce the formation of blood vessels inthe tumor, effectively starving the tumor and/or cause the tumor todrown in its own waste.

Neovascularization: The growth of new blood vessels. Neovascularizationcan be the proliferation of blood vessels in tissue not normallycontaining them, or the proliferation of blood vessels in an ischemic orotherwise damaged tissue. Neovascularization can be pathological when itis unwanted or mediates a pathological process, for example when itoccurs in the retina or cornea.

Passaging cells: Passaging or splitting cells involves transferring asmall number of cells into a new vessel. Cells can be cultured for alonger time if they are split regularly, as it avoids the senescenceassociated with prolonged high cell density. Suspension cultures areeasily passaged with a small amount of culture containing a few cellsdiluted in a larger volume of fresh media. For adherent cultures, cellsfirst need to be detached; which is typically done with a mixture oftrypsin-EDTA. A small number of detached cells can then be used to seeda new culture.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject (such as the inhibition of angiogenesis),alone or in combination with another therapeutic agent(s) orpharmaceutically acceptable carriers. Pharmaceutical agents include, butare not limited to, angiogenic factors, for example bFGF, and VEGF, andanti-angiogenic factors, such as inhibitors of bFGF, or VEGF. Forexample, suitable anti-angiogenic factors include, but are not limitedto, SU5416, which is a specific VEGF-R antagonist, SU6668 which blocksthe receptors for VEGF, bFGF, and PDGF and Avastin®. See, for example,Liu et al., Seminars in Oncology 29 (Suppl 11): 96-103, 2002; Shepherdet al., Lung Cancer 34:S81-S89, 2001. The term pharmaceutical agent alsocan be applied to the bioactive compounds discussed herein, includingspecifically the antiangiogenic compounds listed in TABLE 1, andcharacterized in TABLE 10.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the compositions disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Primary cells: Cells that are cultured directly from a subject. With theexception of some derived from tumors, most primary cell cultures havelimited lifespan. After a certain number of population doublings cellsundergo the process of senescence and stop dividing, while generallyretaining viability.

Signal: A detectable change or impulse in a physical property thatprovides information. In the context of the disclosed methods, examplesinclude electromagnetic signals, such as light, for example light of aparticular quantity or wavelength, for example a wavelength of lightemitted from a fluorescent protein.

Small molecule inhibitor (for example, of an inhibitory ofangiogenesis): A molecule, typically with a molecular weight less than1000 Daltons, or in some embodiments, less than about 500 Daltons,wherein the molecule is capable of inhibiting, to some measurableextent, an activity of some target molecule. In particular embodiments,the small molecule inhibitor is an inhibitor of angiogenesis, whichactivity can be tested, detected, determined, and/or measured usingmethods known in the art and/or described herein.

Test agent: Any agent that is tested for its effects, for example itseffects on a cell. In some embodiments, a test agent is a chemicalcompound, such as an antiangiogenic agent or even an agent with unknownbiological properties.

Therapeutically effective amount/dose: A dose sufficient to have atherapeutic effect, for example to inhibit to some degree advancement,or to cause regression of the disease, or which is capable of relievingsymptoms caused by the disease. For example, a therapeutically effectiveamount of an angiogenesis inhibitor can vary from about 0.1 nM perkilogram (kg) body weight to about 1 μM per kg body weight, such asabout 1 nM to about 500 nM per kg body weight, or about 5 nM to about 50nM per kg body weight. The exact dose of a particulartherapeutic/bioactive compound is readily determined by one of skill inthe art based on the potency of the compound, the age, weight, sex andphysiological condition of the subject, the disease being treated, andso forth.

Treating: Inhibiting the full development of a disease or condition, forexample, in a subject who is at risk for a disease such as cancer.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. The term “ameliorating,” with reference to a disease orpathological condition, refers to any observable beneficial effect ofthe treatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, an improvement in theoverall health or well-being of the subject, or by other parameters wellknown in the art that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Tubule formation potential: The ability of a cell-line to form atube-like structure in vitro, for example a structure similar to a bloodvessel, such as a capillary. Tubule formation potential can bedetermined by determining the pattern displayed by cells which have beeninduced to form tubules, for example by determining the pattern offluorescence from cells expressing fluorescent proteins, such as thecell-lines disclosed herein.

III. Description of Several Embodiments

Described herein is a stringent HTS strategy which, together withadvanced bioinformatics mining tools, was used to identify a new set ofantiangiogenic small molecules. The strategy included the following:

Two cell-based HTS were designed to identify small molecules that blockthe main steps of the angiogenic process: growth of endothelial cellsand tube formation. In both assays, compounds were tested at a low finalconcentration (1 μM) to avoid false positives. The following classes ofbioactive SMs were identified in these assays: 48 growth inhibitors, 35tube formation inhibitors. 11 SMs block both growth and tube formation.3 SMs have been identified as specific inhibitors of tumor growth; thesecompounds are not antiangiogenic but are antitumoral. See TABLE 10 for asummary of the HTS results.

All bioactive compounds were tested for their ability to inducecytotoxicity and apoptosis in endothelial cells. Three out the 77 smallmolecules were cytotoxic. These cytotoxic SMs were not considered for invivo experiments. Another three SM showed 5-8 fold increase in apoptoticpotential, and 11 showed a 2-3 fold increase (see TABLE 10).

Structure similarity analysis has revealed that most of the bioactiveSMs (68) are not structurally related to existing FDA marketedantiangiogenic SMs, SMs currently in clinical trials or SMs annotated asangiogenesis modulators in PubChem, DrugBank, LeadScope, etc. Therefore,based on structure-activity relationship (SAR), the compounds identifiedherein (see TABLE 1) represent a new set of antiangiogenic compoundsfunctionally unrelated to known antiangiogenic SMs. Additional SARanalysis has identified a number of scaffolds which correlate withinhibitory activity of both endothelial cell growth and endothelial celltube formation (see TABLES 2-9).

Structure-activity-relationship (SAR) studies have shown a potentialmechanism of action for some (14) of the SMs of interest. For instance,the structure of some of the growth inhibitors is consistent withtopoisomerase II inhibitory activity while some tube formationinhibitors show structures consistent with known tubulin binders. SeeTABLE 10.

Comparison of the growth inhibitory activity of these compounds inendothelial cells and tumor cells has allowed their classification inseveral groups including: 1) SMs able to inhibit the growth of bothendothelial cells and tumor cells (Compounds 1-73); and 2) SMs whichinhibit the growth of tumor cells but not endothelial cells (Compounds74-77).

Thus, described herein are compounds that exhibit inhibition ofundesirable angiogenesis, and methods for using these compounds to treatangiogenesis-dependent diseases or neoplasms (e.g., solid tumors). Inparticular, the presently disclosed method provides for inhibitingunwanted angiogenesis in a human or animal by administering to the humanor animal with the undesired angiogenesis a composition comprising aneffective amount of at least one of the compounds described herein, suchas specifically one or more of Compounds 1 through 77 as shown inTABLE 1. Examples of such methods involve inhibiting angiogenesis byexposing a tissue or cell mass having the undesirable angiogenesis to anangiogenesis inhibiting amount of one or more compounds, orpharmaceutically acceptable salts or derivatives of such compounds,wherein such compounds are selected from those of Compounds 1 through 77as shown in TABLE 1.

It will be recognized that although the compounds disclosed hereinexhibit antiangiogenic properties, the mechanism for specific action bythe compounds are not necessarily limited to antiangiogenic mechanisms.For example, the compounds may also exhibit cytotoxic properties (thatmay be independent of any antiangiogenic properties) that are useful fortreating neoplasms.

Antiangiogenic drugs are among the most promising agents for theclinical management of cancer and other angiogenesis related diseases,such as endometriosis. A multibillion dollar market has developed overthe past decade involving innumerable pharmaceutical companies that arein the process of developing or attempting to develop antiangiogenicSMs.

Recent advances in the understanding of the angiogenic process haveimpelled the development of a new group of antiangiogenic SMs. Most ofthe antiangiogenic SMs currently being considered in the clinic aretubulin binders or target the tyrosine kinase activity of cell surfacereceptors involved in the angiogenic process such as the VEGF receptor.Some of these compounds (such as inhibitors of the VEGF pathway) havealready shown limited clinical success in the management ofangiogenesis-related diseases, mainly cancer.

In contrast to the antiangiogenic SMs that were previously identified,the innovative approach to identifying antiangiogenic compoundsdescribed herein is not restricted by the subcellular target, rather ittargets the main cellular processes involved in angiogenesis. Therefore,this method has permitted the discovery of novel SM that are not relatedto tyrosine kinase inhibitors, tubulin binders or any other knownantiangiogenic SM currently in development. Additionally this highlystringent screening design guarantees the absence of “false positives,”which commonly represent a major obstacle in HTS.

Therefore, the newly identified SMs provided herein (e.g., Compounds1-77) represent new groups of high quality compound leads, defining newantiangiogenic subcellular targets and opening up the possibility ofdeveloping drugs based on mechanism of action alternative to the onescurrently being considered by academic institutions and privatepharmaceutical industry.

In addition, the screen employed herein has provided information aboutthe specificity of the growth inhibitory activity of some of thebioactive SMs. For instance, some molecules have been found whichinhibit the growth of tumor cells while having no substantial effect onendothelial cells. This enables advanced combinatorial drug regimens.For instance, in the treatment of angiogenesis-dependent tumors it maysometimes be important to deliver first drugs that inhibit tumor growthbut do not affect endothelial cells (which are the main components ofthe vasculature), since drugs are delivered through the tumorvasculature and a functional vasculature is needed for drug delivery.Therefore, drugs like those found in this study with the ability tospecifically inhibit tumor growth, but not endothelial cell growth(e.g., Compounds 74-77) would be of great value for such treatment. Oncethe tumor has been significantly reduced, drugs with inhibitory activityin both tumor cells and endothelial cells might be preferred, since bothtumor cells and endothelial cells need to be targeted. A number of suchdrugs have also been found in this study (for instance, Compounds 1-37and 63-73). Therefore, the SM specificity information obtained in thisstudy will be very useful in the development of anticancer SM therapies.

The bioactive SMs identified herein represent candidates to be appliedto the clinical management of a variety of angiogenesis related diseasesincluding (but not limited to) cancer, endometriosis, diabeticretinopathy, age-related macular degeneration, etc.

Thus, disclosed herein are pharmaceuticals composition for treating anangiogenesis-dependent disease, comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically-acceptable salt thereof. In particular examples, thepharmaceutical compositions further comprising[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof. In other examples, theangiogenesis-dependent disease comprises cancer, retinopathy,endometriosis, arthritis, or psoriasis. In further examples, thecomposition is administered topically, intravenously, orally,parenterally, or as an implant. In still other examples, thepharmaceutical composition further comprises an additional angiogenesisinhibitor.

Also disclosed herein are pharmaceutical compositions for inhibitingaberrant angiogenesis, comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically-acceptable salt thereof. In some examples, thepharmaceutical compositions further comprise[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof. In some examples, the aberrantangiogenesis is stimulated by a tumor, which can be benign or malignant.

Also disclosed herein are pharmaceutical compositions for inhibitinggrowth of neoplastic tissue, comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically-acceptable salt thereof. In particular examples, thepharmaceutical compositions further comprise[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof.

Additionally disclosed are methods of treating an angiogenesis-dependentdisease, comprising: administering to a subject having or predisposed toan angiogenesis-dependent disease a therapeutically effective amount ofa composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. In particular examples, thecomposition further comprises [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof. Infurther examples, the angiogenesis-dependent disease comprises cancer,retinopathy, endometriosis, arthritis, or psoriasis. In some examples,the composition is administered topically, intravenously, orally,parenterally, or as an implant. In still other examples, the methodsfurther comprise administering to the subject an additional angiogenesisinhibitor, such as an inhibitor of bFGF, FGF, or VEGF.

Also disclosed herein are methods of inhibiting undesired angiogenesisin a subject, comprising: identifying a subject wherein angiogenesis isnot desired, and administering to the subject a therapeuticallyeffective amount of a composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. In particular examples, thecomposition further comprises [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof. Inother examples, the method further comprises administering an additionalinhibitor of angiogenesis, such as an inhibitor of bFGF, FGF, or VEGF.In some examples, the undesired angiogenesis comprises tumorangiogenesis, for example wherein the tumor is benign or malignant.

Further disclosed herein are methods of inhibiting a neoplasm in asubject, comprising: administering to the subject a therapeuticallyeffective amount of a composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. In particular examples, thecomposition further comprises [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof.

Lastly, disclosed herein are methods of inhibiting angiogenesis in atissue or a target area in a subject wherein the formation of new bloodvessels is not desired, comprising identifying a tissue or target areain a subject wherein the formation of new blood vessels is not desired;and

introducing directly or indirectly into the tissue or target area aneffective amount of a composition comprising at least one of2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methyl propanoate(NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC 122657),maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof, thereby inhibitingangiogenesis in the tissue or target area. In particular examples, thecomposition further comprises [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof. In someexamples, the target area comprises skin, a tumor, a retina, a joint, orendometrial tissue. In other examples, the subject has or is predisposedto developing a tumor, retinopathy, endometriosis, arthritis, orpsoriasis.

IV. Bioactive Molecules

A set of diverse chemical compounds (Diversity Set I) was obtained fromthe NCI/DTP Open Chemical Repository (available on-line atdtp.cancer.gov). Diversity Set I included 1990 compounds, each of whichcontains at least five new pharmacophores and five or fewer rotatablebonds as determined by the Chem-X program (Oxford Molecular Group,Oxford, UK). Although Diversity Set I is no longer available as a set,individual compounds can be obtained from the NIC/DTP Open ChemicalRepository (though large amounts of NSC 675865, NSC 18877, NSC 176327,NSC 521777, NSC 166687, and NSC 119889 are not available). It will beunderstood by one of skill in the art that each member of Diversity SetI represents a large family of molecules, information related to whichcan be accessed through public databases. With the identification hereinof the antiangiogenic activity of representative Compounds 1-77, methodsof testing each related class of molecules for antiangiogenic activityare now enabled, as is use of any molecules identified through suchscreening as antiangiogenic agents. Structure similarity analyses,including structure-activity relationship analysis as described below inSection V, will be used to identify additional compounds that couldpotentially show antiangiogenic activity. Such analysis will enable thescreening of compounds that are not associated with the Diversity Set Ifamilies, but are available through public databases.

The compounds in Diversity Set I were screened for potentialantiangiogenic activity, as provided herein. Table 1 includes 77compounds from Diversity Set I that were discovered to inhibit (1)endothelial cell growth, (2) tube formation, (3) endothelial cell growthand tube formation, or (4) specific tumor cell growth withoutconcomitant endothelial cell growth inhibition. Specifically, compounds1-37 are endothelial cell growth inhibitors, compounds 38-62 are tubeformation inhibitors, compounds 63-73 are both endothelial cell growthand tube formation inhibitors, and compounds 74-77 are specific tumorcell growth inhibitors. Of the 77 compounds found to be bioactive in thescreening assays, compounds 63, 64, 67, 69, and 71 are considered to beof particular interest. Each of these compounds inhibited bothendothelial cell growth and tube formation without cytotoxicity (lessthan 10% cytotoxicity is considered to be a basal level).

TABLE 1 BIOACTIVE SMALL MOLECULES Com- pound Structure NSCNumber/Nomenclature  1

NSC 329226 4-methoxy-2-nitrobenzo[e][1]benzofuran  2

NSC 15234 (E)-N-(9H-fluoren-2-yl)-3-phenylprop-2- en-1-imine  3

NSC 15226 N-(9H-fluoren-2-yl)-1-pyridin-2- ylmethanimine  4

NSC 24076 diethylaminomethoxymethanedithioic acid; sodium  5

NSC 26081 3-(dimethylamino)-1-(4- methoxynaphthalen-1-yl)propan-1-onechloride  6

NSC 133896 2,3-dinitrofluoren-9-one  7

NSC 675865 2-(7-amino-[1,2]thiazolo[4,5-d]pyrimidin-3-yl)-5-(hydroxymethyl)oxolane-3,4-diol  8

NSC 10460 [4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-dien-1-ylidene)methyl]phenyl]azanium chloride  9

NSC 207895 7-(4-methylpiperazin-1-yl)-4-nitro-1-oxido-2,1,3-benzoxadiazol-1-ium 10

NSC 99445 [5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate 11

NSC 88903 6,6-dimethyl-1-(4-phenylbutyl)-1,3,5- triazine-2,4-diaminehydrochloride 12

NSC 177407 5,6-dichloro-2-[3-(trifluoromethyl)phenyl]-1H-imidazo[4,5-b]pyrazine 13

NSC 123111 (6-Methoxy-1,5-dimethyl-4,7-dioxo-1,1a,2,4,7,8b-hexahydroazireno[2′,3′:3,4]-pyrrolo[1,2-a]indol-8-yl)methyl carbamate 14

NSC 329261 3-(furan-2-ylmethyl)-5-(pyridin-3-ylmethylidene)-2-sulfanylidene-1,3- thiazolidin-4-one (The crossed bondrepresents a double bond for which the cis- or trans- structure isunknown or can be either.) 15

NSC 13316 [2-(4-chlorophenyl)quinolin-4-yl]-piperidin- 2-ylmethanol 16

NSC 5844 N-N′-bis(7-chloroquinolin-4-yl)ethane-1,2- diamine 17

NSC 5857 6-tert-butyl-2-[(3-tert-butyl-5-chloro-2-hydroxy-6-methylphenyl)methyl]-4-chloro- 3-methylphenol 18

NSC 656202 [(1R,2R,3E,7S,11E,13S,15S)-2-hydroxy-7- methyl-5-oxo-6-oxabicyclo[11.3.0]hexadeca-3,11-dien-15- yl] 2-(dimethylamino)acetate 19

NSC 2186 3,3-bis(4-hydroxy-2-methyl-5-propan-2-ylphenyl)-2-benzofuran-1-one 20

NSC 97845 (16E)-16-hydroxyimino-13-methyl-3-(2-methylprop-2-enoxy)-6,7,8,9,11,12,14,15-octahydrocyclopenta[a]phenanthren-17-one 21

NSC 368891 6,6-dimethyl-1-[3-(phenylsulfanylmethyl)-phenyl]-1,3,5-triazine-2,4-diamine hydrochloride 22

NSC 126710 5-(3,4-dichlorophenyl)-6-[(3-methyl-4-nitrophenoxy)methyl]pyrimidine-2,4- diamine 23

NSC 109836 6,6-dimethyl-1-[3-[(3- nitrophenyl)methoxy]-phenyl]-1,3,5-triazine-2,4-diamine chloride 24

NSC 67485 2-tert-butyl-4-[1-(5-tert-butyl-4-hydroxy-2-methylphenyl)butyl]-5-methylphenol 25

NSC 47932 4-chloro-2,6-bis[(5-chloro-2- hydroxyphenyl)methyl]phenol 26

NSC 176327 3-(9-methoxy-5,11-dimethylpyrido[4,3-b]carbazol-2-ium-6-yl)propan-1-amine chloride 27

NSC 48630 1-[(3S,8R,9S,10R,13S,14S,17R)-3,17- dihydroxy-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16- dodecahydrocyclopenta[a]phenanthren-17-yl]-3-(dimethylamino)propan-1-one chloride 28

NSC 71669 5-[3-(trifluoromethyl)phenyl]-6-[[3-(trifluoromethyl)phenyl]methyl]pyrimidine- 2,4-diamine 29

NSC 150289 2,3-dibromo-1-(4-nitrophenyl)-3-quinolin- 4-ylpropan-1-one 30

NSC 311153 5,11-dimethyl-2-(2-piperidin-1-ylethyl)-6H-pyrido[4,3-b]carbazol-2-ium-9-ol acetate 31

NSC 45238 [(3S,8R,9S,10R,13S,14S,16E)-16-(1-acetyloxy-2,2,2-trifluoroethylidene)-10,13-dimethyl-17-oxo-2,3,4,7,8,9,11,12,14,15-decahydro-1H-cyclopenta[a]phenanthren-3- yl] acetate 32

NSC 156305 N-[4-(acridin-9-ylamino)phenyl]- methanesulfonamide;methanesulfonic acid 33

NSC 62914 2-tert-butyl-6-[[3-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-2-hydroxy-5- methylphenyl]methyl]-4-methylphenol34

NSC 606985 4-Ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2- b]quinolin-4-yl aminoacetatehydrochloride 35

NSC 254681 (1S,3S)-3-acetyl-11-amino-1-(4-amino-5-hydroxy-6-methyloxan-2-yl)oxy-3,6- dihydroxy-10-methoxy-2,4-dihydro-1H-tetracene-5,12-dione hydrochloride 36

NSC 268665 4-amino-1-[6-(hydroxymethyl)-2,2-diphenyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3,2]dioxastannol-4-yl]pyrimidin-2-one 37

NSC 306698 3-[(2,4- dimethylphenyl)carbamoyl]naphthalen-2- olate;N-(2,4-dimethylphenyl)-3- hydroxynaphthalene-2-carboxamide; nickel(2+)38

NSC 4972 2-tert-butylbenzene-1,4-diol 39

NSC 19630 (2,5-dioxopyrrol-1-yl)methyl propanoate 40

NSC 2805 2-(2,5-dihydroxy-4-methylphenyl)-5- methylbenzene-1,4-diol 41

NSC 16555 2-(4-methylphenyl)sulfonyloxyacetic acid 42

NSC 3535 [(1S,4R,6R)-1,7,7-trimethyl-6- bicyclo[2.2.1]heptanyl] 2-thiocyanatoacetate 43

NSC 27063 [(E)-(4- methylsulfonylphenyl)methylideneamino] thiourea 44

NSC 47924 1-[(4-methoxyanilino)methyl]naphthalen-2- ol 45

NSC 36738 S-[2-oxo-2-(quinolin-6-ylamino)ethyl] carbamothioate 46

NSC 108895 1-(1,3-benzodioxol-5-ylmethyl)pyrrolidine- 2-carboxylic acid47

NSC 681152 methyl 4-[2-(2,5- dihydroxyphenyl)ethyl]benzoate 48

NSC 632536 4-N-phenyl-1-N-(2,2,6,6-tetramethylpiperidin-4-yl)benzene-1,4- diamine 49

NSC 122351 azidobenzene; 1H-indene; prop-1-en-2- ylbenzene 50

NSC 268879 (2-aminopurin-9-id-6-yl)sulfanyl- hexylmercury 51

NSC 48458 3,8-dibenzyl-5,10-ditert-butyl-2,4,7,9-tetrahydro-[1,3]oxazino[6,5- g][1,3]benzoxazine 52

NSC 209910 3,5-di(cyclododecyl)-1,3,5-thiadiazinane-2- thione 53

NSC 328087 2-(3,5-dichloro-2-hydroxyphenyl)-3-(2-pyridin-2-ylethyl)-1,2-dihydroquinazolin-4- one 54

NSC 521777 17-[(E)-2,6-dihydroxy-6-methyl-3-oxohept-4-en-2-yl]-2,16-dihydroxy-4,4,9,13,14-pentamethyl-8,10,12,15,16,17-hexahydro-7H-cyclopenta[a]phenanthrene-3,11-dione 55

NSC 310551 copper; [(6-methylpyridin-2- yl)methylidene-amino]-[methylsulfanyl(sulfoniumylidene)- methyl]azanide 56

NSC 292222 Maytansinol isobutyrate or 4,24-Dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5] hexacosane, maytansine deriv.57

NSC 321237 Mercury, (4-aminophenyl)(6- thioguanosinato-N7,S6)- 58

NSC 259969 Deoxybouvardin or 24-hydroxy-10-(4-methoxybenzyl)-4,7,9,13,15,29- hexamethyl-22-oxa-3,6,9,12,15,29-hexaazatetracyclo- [14.12.2.2~18,21~.1~23,27~]tritriaconta-18,20,23(31),24,26,32-hexaene- 2,5,8,11,14,30-hexone 59

NSC 259968 Bouvardin or 17,24-dihydroxy-10-(4-methoxybenzyl)-4,7,9,13,15,29- hexamethyl-22-oxa-3,6,9,12,15,29-hexaazatetracyclo- [14.12.2.2~18,21~.1~23,27~]tritriaconta-18,20,23(31),24,26,32-hexaene- 2,5,8,11,14,30-hexone 60

NSC 203328 3-bromo-4-[(2-bromo-6-tert-butyl-4-cyanophenoxy)-phenylphosphoryl]oxy-5- tert-butylbenzonitrile 61

NSC 166687 zinc; 8-hydroxy-3,4,4a,5,6,7,8,8a-octahydro-2H-quinolin-1-ide-5-sulfonic acid;5-sulfo-1,2-dihydroquinolin-8-olate 62

NSC 119889 2,3,4,5-tetrabromo-6-(3,6-dihydroxy-9H- xanthen-9-yl)benzoicacid 63

NSC 676693 3-(4-methoxyphenyl)thieno[2,3- b]pyrrolizin-8-one 64

NSC 122657 1-benzylsulfonyl-2,4-dinitrobenzene 65

NSC 295642 [benzylsulfanyl(sulfoniumylidene)methyl]-(1-pyridin-2-ylethylideneamino)azanide; chlorocopper 66

NSC 13480 (2-phenylbenzo[h]quinolin-4-yl)-piperidin- 2-ylmethanol 67

NSC 150117 2-benzylidene-3-(cyclohexylamino)-3H- inden-1-one chloride 68

NSC 18877 [4-(1-benzothiophene-3-carbonyl)-2-oxide-1,2,5-oxadiazol-2-ium-3-yl]-(1- benzothiophen-3-yl)methanone 69

NSC 48300 [4-[(4-arsonophenyl)methyl]phenyl]arsonic acid 70

NSC 321206 bromocopper; (dipyridin-2- ylmethylideneamino)-[methylsulfanyl(sulfoniumylidene)methyl] azanide 71

NSC 292596 chloroplatinum(1+); 2-(4-methylpiperidin-1-yl)ethanethiolate; dihydrate 72

NSC 112200 2,5-dibromo-3,6-dimethylbenzene-1,4-diol;2,5-dibromo-3,6-dimethylcyclohexa-2,5- diene-1,4-dione 73

NSC 274547 gold(1+); 1-(2,3,4-triaza-5-azanidacyclopenta-1,3-dien-1-yl)-2,3,4-triaza-5-azanidacyclopenta-1,3-diene; triphenylphosphane 74

NSC 4265 1,10-phenanthroline hydrochloride 75

NSC 130830 5-[(benzylamino)methyl]quinolin-8-ol 76

NSC 54044 N′-[1-(1-oxonaphthalen-2-ylidene)ethyl]pyridine-4-carbohydrazide 77

NSC 327705 2-chloro-3-phenanthro[9,10-e]-[1,2,4]triazin-3-ylsulfanylnaphthalene-1,4- dione

V. Potentially Active Related Structures

Additional potentially active compounds can be predicted by comparingthe structure of any of Compounds 1-77 to the structures of other knowncompounds (or newly-derived compounds) and determining the similaritythrough structure-activity relationship (SAR) or quantitativestructure-activity relationship (QSAR) analysis. There are several knownmethods for SAR and QSAR analysis, including Tanimoto's algorithm(Dogra, “Script for computing Tanimoto coefficient,” QSARWorld,available on-line at qsarworld.com/virtual-workshop.php, Jul. 5, 2007)and LeadScope's clustering algorithm (Leadscope Inc., Columbus, Ohio).SAR and QSAR methods compare molecular structures and determinestructural features shared by the molecules.

Because Compounds 1-77 (shown in TABLE 1) were derived from a diversityset of small molecules, most of the active compounds are determined notto be structurally related when using Tanimoto's algorithm. However,clustering using LeadScope's algorithm has been used successfully todetermine structural similarities between some of the compounds in TABLE1 and other known compounds. For instance, many compounds in DiversitySet I have family members (which are related by structure) that are notincluded in Diversity Set I. Such family members and other compounds canbe evaluated using LeadScope's algorithm, which searches for (1) large,commonly occurring substructures; (2) substructures that discriminatefor a biological response; or (3) substructures that discriminate formembership in a set of compounds (Cross et al., J. Med. Chem.,46:4770-4775, 2003).

Two prediction models were built based on bioactive Compounds 1-77: onefor compounds that inhibit endothelial cell growth and another forcompounds that inhibit tube formation. The models were built usingLeadScope software, and were based on logistic regression. To develop apredictive model for small molecules that inhibit growth of endothelialcells, a total of 70 compounds with known growth inhibitory activity and76 compounds with no inhibitory effect were used. The predictive modelaccurately predicted 70/70 positive compounds and accurately predicted76/77 negative compounds, producing a concordance of 99.3% with 98.6%sensitivity and 100.0% specificity. Concordance is a measure of theoverall model accuracy, i.e., 76/77=99.3%. Sensitivity is a measure ofhow well the model predicts true positives. Sensitivity is calculatedusing the equation: sensitivity=TP/(TP+FN), where TP is the number oftrue positives and FN is the number of false negatives. Specificity is ameasure of how well the model predicts true negatives. Specificity iscalculated using the equation: specificity=TN/(TN+FP), where TN is thenumber of true negatives and FP is the number of false positives. Theconcordance for a test set of molecules was 98.0%, with 69 truepositives, 1 false positive, 75 true negatives, and 2 false negatives.The sensitivity was 97.2% with 98.7% specificity. Each molecule in thetraining and test sets was tested in an endothelial cell growth assay toassess the accuracy of the prediction.

A predictive model for small molecules that inhibit tube formation wasalso developed. Concordance for the training set was 100.0%, with 100.0%sensitivity and specificity. A test set of molecules produced 71.0%concordance, with 12 true positives, 8 false positives, 32 truenegatives, and 10 false negatives. The sensitivity was 54.5% with 80.0%specificity. Each molecule in the training and test sets was tested in atube formation assay to assess the accuracy of the prediction.

These predictive models were applied to various databases, including theNCI small molecule database (dtp.nci.nih.gov/), DrugBank (drugbank.ca),LeadScope (leadscope.com), and PubChem (pubchem.ncbi.nlm.nih.gov/).Based on the models, several hundred small molecules have been predictedto inhibit endothelial cell growth and tube formation. For example,several substructures (“scaffolds”) were identified in Compounds 1-77that are predicted to produce endothelial cell growth inhibition or tubeformation inhibition.

The scaffolds were identified using LeadScope software. The softwarecalculates a “z-score” for each compound and activity. The z-scorecompares the mean activity of a subset to its expected value:

$z = {\left( {{\overset{\_}{x}}_{1} - {\overset{\_}{x}}_{0}} \right)\sqrt{\frac{n_{1}n_{0}}{s_{0}^{2}\left( {n_{0} - n_{1}} \right)}}}$(Cross et al., J. Med. Chem., 46:4770-4775, 2003) After performingclustering analysis, each cluster contains a plurality of smallmolecules having a common scaffold, each molecule having a z-scorevalue. A low z-score value indicates low values for the definedactivity, e.g., growth or tube formation. Thus, a small molecule with alow z-score for growth results in less growth than a molecule with ahigher z-score. An average z-score value is associated with the cluster,based on the individual z-score values of the small molecules. A clusterwith a low z-score value is likely to contain small molecules withindividual low z-score values. In the present embodiments, onlyscaffolds included in clusters with an average z-score of less than −2were considered. For example, molecules in clusters having an averagegrowth z-score of less than −2 are predicted to be potential growthinhibitors. These identified scaffolds have predictive value for bothgrowth inhibition and/or tube formation inhibition.

Several representative clusters of molecules having scaffolds predictedto inhibit endothelial cell growth are shown below in TABLES 2-6.Additional molecule clusters having scaffolds predicted to inhibit tubeformation are shown in TABLES 7-9.

TABLE 2 CLUSTER 368

NSC Number/ Compound Structure Nomenclature 55

NSC 310551 copper; [(6- methylpyridin-2-yl) methylidene-amino]-[methylsulfanyl (sulfoniumylidene)- methyl]azanide 65

NSC 295642 [benzylsulfanyl (sulfoniumylidene) methyl]-(1-pyridin-2-ylethylideneamino) azanide; chlorocopper 70

NSC321206 bromocopper; (dipyridin-2-yl- methylideneamino)-[methylsulfanyl- (sulfoniumylidene) methyl]azanide

TABLE 3 CLUSTER 71

NSC Number/ Compound Structure Nomenclature 30

NSC 311153 5,11-dimethyl- 2-(2-piperidin- 1-ylethyl)-6H- pyrido[4,3-b]carbazol-2-ium- 9-ol acetate 30-A

NSC 163443 2-(5,11- dimethylpyrido [4,3-b]carbazol- 6-yl)ethyl benzoate30-B

NSC 359449 2-(5,11- dimethyl- 6H-pyrido [4,3-b] carbazol-2- ium-2-yl)-N,N-diethyl- ethanamine acetate 30-C

NSC 311152 2-(2-diethyl- aminoethyl)- 5,11-dimethyl- 6H-pyrido [4,3-b]carbazol-2- ium-9-ol acetate 30-D

NSC 176327 3-(9-methoxy- 5,11- dimethylpyrido [4,3-b]carbazol-6-yl)propan- 1-amine (Same NSC # as 26, but not the chloride salt.)

TABLE 4 CLUSTER 358

NSC Number/ Compound Structure Nomenclature 34

NSC 606985 4-Ethyl-3,14- dioxo- 3,4,12,14- tetrahydro-1H-pyrano[3′,4′:6,7] indolizino[l,2-b] quinolin-4-yl aminoacetatehydrochloride 34-A

NSC 100880 sodium; hydride; 2-hydroxy-2-[8- (hydroxy- methyl)-9-oxo-11H- indolizino[1,2-b] quinolin-7-yl] butanoic acid 34-B

NSC 609699 Topotecan, Hycamtin, or 10-((di- methylamino) methyl)-4-ethyl-4,9- dihydroxy- 1H-pyrano [3′,4′:6,7]- indolizino[1,2-b]quinoline- 3,14(4H,12H)- dione hydrochloride 34-C

NSC 639174 2-amino-N-(4- ethyl-4-hydroxy- 3,14-dioxo- 3,4,12,14-tetrahydro-1H- pyrano[3′,4′:6,7] indolizino [1,2-b] quinolin-10-yl)acetamide hydrochloride

TABLE 5 CLUSTER 337

NSC Number/ Compound Structure Nomenclature 17

NSC 5857 6-tert-butyl-2- [(3-tert-butyl-5- chloro-2-hydroxy-6-methylphenyl) methyl]-4-chloro- 3-methylphenol 25

NSC 47932 4-chloro-2,6-bis [(5-chloro-2- hydroxyphenyl) methyl]phenol

TABLE 6 CLUSTER 479

NSC Number/ Compound Structure Nomenclature 64

NSC 122657 1-benzylsulfonyl- 2,4-dinitrobenzene 64-A

NSC 228148 7-benzylsulfonyl-4- nitro-2,1,3- benzoxadiazole

TABLE 7

NSC Number/ Compound Structure Nomenclature 50

NSC 268879 (2-aminopurin- 9-id-6- yl)sulfanyl- hexylmercury 57

NSC 321237 Mercury, (4-aminophenyl) (6-thioguanosinato- N7, S6)-

TABLE 8

NSC Number/ Compound Structure Nomenclature 58

NSC 259969 Deoxybouvardin or 24-hydroxy-10-(4-methoxybenzyl)-4,7,9,13,15,29- hexamethyl-22-oxa-3,6,9,12,15,29-hexaazatetracyclo- [14.12.2.2~18,21~.1~23,27~]tritriaconta-18,20,23(31),24,26,32- hexaene-2,5,8,11,14,30-hexone 59

NSC 259968 Bouvardin or 17,24-dihydroxy-10-(4-methoxybenzyl)-4,7,9,13,15,29- hexamethyl-22-oxa-3,6,9,12,15,29-hexaazatetracyclo- [14.12.2.2~18,21~.1~23,27~]tritriaconta-18,20,23(31),24,26,32- hexaene-2,5,8,11,14,30-hexone

TABLE 9

NSC Number/ Compound Structure Nomenclature 38

NSC 4972 2-tert-butylbenzene- 1,4-diol 40

NSC 2805 2-(2,5-dihydroxy-4- methylphenyl)-5- methylbenzene- 1,4-diol 47

NSC 681152 methyl 4-[2-(2,5- dihydroxyphenyl) ethyl]benzoate

VI. Bioactivity Tests

Once a new compound has been identified as a potential antiangiogeniccompound based on comparison to one of Compounds 1-77, or the compoundsadditionally listed in any of TABLES 2-9, or any derivative thereof, theidentified potential therapeutic compound can be tested for bioactivity.By way of example, any of the methods described herein can be used. Thefollowing list provides a description of representative but non-limitingexample bioactivity assays. Additional assays will be known to those ofordinary skill; for instance, additional assays are described in U.S.application Ser. No. 12/060,752 (published as US 2009/0088341 on Apr. 2,2009; incorporated herein by reference in its entirety).

i. Fluorescence-Based Growth Assay

A real time growth assay has been applied to mono- or multiple-cellcultures (co-culture). The fluorescence signal emitted by a culture ofthe disclosed fluorescent cell-lines is proportional to the number offluorescent cells present in the culture. In other words, thefluorescence signal, for example measured as the intensity of theemission maxima, from a population of fluorescent cells of one type in aculture will double as the number of fluorescent cells of that type inthe culture doubles. Conversely, the fluorescence signal, for examplemeasured as the intensity of the emission maxima, from a population ofcells of one type in a culture will be reduced to half if the number ofcells of that type in the culture is divided in half. These propertiescan be used to measure the effect of an exogenous agent, such as one ormore additional cell-lines, or a test agent (such as a bioactive SM), onthe fluorescent cells in culture. At some point the total fluorescenceof a culture may reach signal saturation, such that the signal reaches aplateau as a function of cell number. The effect of an additionalcell-line (for example a different cell-line) on a first fluorescentcell-line can be determined (this can be extended to multiple cell-linesand even one or more fluorescent cell-lines, or combinations thereof,for example in a multiplex assay or 3-dimensional co-culture).

The difference between the fluorescence signal (such as the intensity ofthe fluorescence signal at a particular wavelength, for example theemission maxima of the fluorescence signal) attributable to thefluorescent cell-line of interest grown in co-culture with one or moreadditional cell-lines relative to a control in some instances will be atleast about 10%, meaning that the growth rate of the cell-line ofinterest is either reduced or increased by at least about 10%, such asat least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.The difference may be a statistically significant difference. Thus, thepresence of a bioactive SM with or without one or more additionalcell-lines can induce a statistically significant difference in thegrowth rate of a fluorescent cell-line of interest, as compared to thecontrol, such as a value indicative of the basal rate of growth of thefluorescent cell-line, or the fluorescent cell-line of interest grown inthe absence of the SM and/or other cells or cell-lines, for examplegrown in mono-culture. The at least one SM and/or additional cell-line(or additional cell-lines) will in some instances have a negative impacton the first fluorescent cell-line, such that the number of cells of thefirst fluorescent cell-line is reduced as a function of time relative toa control. In other examples, the at least one SM and/or additionalcell-line (or additional cell-lines) will have a positive impact on thefirst fluorescent cell-line, such that the number of cells of the firstfluorescent cell-line present in a cell culture increases as a functionof time relative to a control. It is also contemplated that thefluorescent cell-line of interest can be co-cultured with primary cells,such as primary cells obtained from a subject, for example tumor cells,and the effect of the primary cells on the growth rate of thefluorescent cell-line of interest determined. Such co-cultures may beestablished in 2 or 3 dimensions.

The effect of each fluorescent cell-line on other fluorescentcell-line(s) present in co-culture can be determined, for example in amultiplex assay. For example, using appropriate filters or FACS analysisamong other techniques, fluorescent cell-lines expressing differentfluorescent proteins, such as red, green, yellow, cyan and the likefluorescent proteins can be discriminated and the fluorescent signalattributable to the different cell-lines determined. Thus, the growthrates of individual fluorescent cell-lines can be determined from amono-culture and/or a co-culture of two or more fluorescent cell-lines.Such analysis greatly enhances the information that can be obtainedabout the individual fluorescent cell-lines.

As described herein, in addition to determining the effect of cell-lineson a fluorescent cell-line of interest, the growth assays can be used todetermine if an exogenous agent, such as a test agent, for example achemical agent (for instance a derivative of one of Compounds 1-77, or astructurally related compound), affects the growth of a fluorescentcell-line of interest. This can also be extended to multiple cell-lines(either fluorescent or not grown in co-culture, for example in amultiplex assay). For instance, the growth assay can be used todetermine if an exogenous agent, such as a test agent (for example apotential modulator of angiogenesis, such as a potential inhibitor ofangiogenesis such as one of Compounds 1-77 or derivatives thereof),growth factor, patient sample, etc. affects the growth rate of afluorescent cell-line of interest, such as one or more of thefluorescent cell-lines disclosed herein. In addition, the differentialeffect of the exogenous agent on the different cell-lines can bedetermined, as can the combinatorial effect of the exogenous agent andthe cells on a cell-line of interest.

A fluorescent cell-line of interest can be contacted with an exogenousagent and the impact of the exogenous agent on the growth of thefluorescent cell-line of interest can be determined. For example, adifference between the fluorescence signal of the fluorescent cell-lineof interest and a control indicates that the exogenous agent, such as atest agent (for example a potential modulator of angiogenesis, such as apotential inhibitor of angiogenesis), growth factor, patient sample,different cell-line, etc. is a modulator (such as an inhibitor) ofangiogenesis. Thus, in several embodiments, one or more of the disclosedfluorescent cell-lines growing in culture are contacted with a testagent (or test agents) to determine if the test agent is a modulator ofangiogenesis. Exemplary test agents include compounds that arestructurally related to any one of Compounds 1-77 (TABLE 1),structurally related to the scaffolds presented in any one of TABLES2-9, derivatives or fragments of any of the compounds described herein,and so forth. Following contact with the test agent, the fluorescence ofthe culture can be measured versus time and/or concentration todetermine the impact of the exogenous agent on the one or morefluorescent cell-lines present in the culture. For example, thefluorescence signal generated by a fluorescent cell-line of interest(such as the intensity of the fluorescence signal at a particularwavelength, for example the emission maxima of the fluorescence signal)can be measured to determine if the fluorescence signal attributable tothe fluorescent cell-line of interest (such as the intensity of thefluorescence signal at a particular wavelength, for example the emissionmaxima of the fluorescence signal) is increasing as a function ofconcentration of the exogenous agent, time, or both, for example bycomparison with a control, such as a value indicative of the basal rateof growth of the fluorescent cell-line of interest or the fluorescentcell-line of interest not contacted with the exogenous agent. In severalembodiments, the control is a known value indicative of normal growth ofthe fluorescent cell-line of interest, for example the doubling time ofcellular number. In some embodiments, the control is the fluorescencesignal of a culture of cells (typically, but not necessarily, a cultureof the fluorescent cell-line of interest) not contacted with theexogenous agent.

In some embodiments, an exogenous agent, such as a test agent, decreasesthe growth rate of the fluorescent cell-line of interest. A test agentexhibiting such an activity is identified as a potential inhibitor ofangiogenesis (that is, having antiangiogenic activity) and would be ofuse in treating a disease or condition in which normal angiogenesis isincreased, for example cancer. In some embodiments, a decrease in thegrowth rate of the fluorescent cell-line of interest relative to acontrol is at least about a 30%, at least about a 40%, at least about a50%, at least about a 60%, at least about a 70%, at least about a 80%,at least about a 90%, at least about a 100%, at least about a 150%, atleast about a 200%, at least about a 250%, at least about a 300%, atleast about a 350%, at least about a 400%, at least about a 500%decrease. Because the fluorescence signal attributable to a fluorescentcell-line of interest is proportional to the number of cells of thecell-line of interest present, the percentage decrease can be measuredas a percentage decrease in the fluorescent signal, for example thefluorescence intensity at a particular wavelength, such as the emissionmaxima, attributable to the cell-line of interest. In additionalembodiments, the decrease is a statistically significant decrease ascompared to a control.

ii. Fluorescence-Based Tubule Formation Assay

Cultures of fluorescent cell-lines expressing different fluorescentproteins, such as the fluorescent cell-lines disclosed herein and inU.S. application Ser. No. 12/060,752 (published as US 2009/0088341 onApr. 2, 2009; incorporated herein by reference in its entirety) can beapplied to tubule formation assays. Formation of new blood vessels isfundamental to angiogenesis and is the focus of many drug screening andcell signaling studies. Blood vessel development is a significant eventin the development and growth of solid tumors, and is involved in woundhealing, retinopathy and macular degeneration. Fluorescent cell-lines,and in particular the disclosed endothelial fluorescent cell-lines, areideal for use in assays for assessing the degree of blood vesselformation using in vitro cell culture assays (see for example Auerbachet al. Clinical Chemistry 49:1, 32-40, 2003; Taraboletti and Giavazzi,EJC 40, 881-889, 2004). Because no fluorescent/colorimetric staining isneeded, the tubule formation assay can be followed over time and can bedirectly visualized used in existing instrumentation, such as the BDPathway™ Bioimager (BD Bioscience, San Jose, Calif.). This allows forthe study of the interaction between different cells types, or between aSM(s) of interest and/or one or more cell types, in this angiogenesis invitro assay. In addition, the effects of SMs on tubule formationpotential can also be determined for a co-culture of a fluorescentcell-line of interest with primary cells, such as primary cells obtainedfrom a subject, for example tumor cells. Such co-cultures can beestablished as 2-dimensional or 3-dimensional co-cultures, such as thosedescribed in U.S. patent application Ser. No. 12/802,666.

Tubule formation assays are typically based on the ability ofendothelial cells, such as fluorescent endothelial cells(stably-transfected to express a fluorescent protein), to form distinctblood-vessel-like tubules in an extracellular matrix (such as BDMatrigel™ Matrix available from BD Bioscience, BME available fromTrevigen, or GELTREX™ available from Invitrogen®, and the like). Thecells are visualized by microscopy, such as fluorescence microscopy inthe case of fluorescent cells, and the ability of one or more compoundsof interest to affect the ability of a fluorescent cell-line of interestto form tubules (also called the tubule formation potential) isdetermined. The determination of tubule formation can be performed bymanual tracing or by automated confocal imaging system, for exampleusing a BD Pathway™ Bioimager in conjunction with AngioApplication™.Using fluorescent cell-lines, tubule formation assays can be performedon live cells, for example to avoid artifacts that may arise fromfixation of cells, such as the disruption of tubules. Several parameterscan be measured in tubule formation assays, such as the total area ofthe tubules, the total number of tubules, number of nodes, number ofbranch points, the number of tubes per node, and/or node area. In someembodiments, the tubule formation potential is determined by a computerimplemented method, for example using the program AngioApplication™.

Fluorescent cell-lines can be used to determine the effects of anexogenous agent, such as cell-lines and test agents, on tubuleformation. In particular examples, a test agent is one or more compoundsthat are structurally related to any one of Compounds 1-77 (TABLE 1),structurally related to the scaffolds presented in any one of TABLES2-9, derivatives or fragments of any of the compounds described herein,and so forth. In some embodiments, multiple fluorescent cell-lines aregrown in co-culture. Thus, the effect of each fluorescent cell-line onthe other fluorescent cell-line(s) present can be determined, or thedifferential effect of an exogenous agent, such as a test agent, orpatient sample, on the different cell-lines can be assessed in amultiplex assay. For example using appropriate filters, the fluorescentsignal from fluorescent cell-lines expressing different fluorescentproteins, such as red, green, yellow, cyan fluorescent proteins can bediscriminated and the fluorescent signal attributable from the differentfluorescent cell-lines determined. Thus, the tubule formation potentialof individual cell-lines can be determined from a mono-culture or even aco-culture, for example a co-culture of more than one fluorescentcell-line.

When grown in co-culture, a difference between the tubule formationpotential of the fluorescent cell-line of interest from a control, sucha mono-culture of the fluorescent cell-line of interest indicates thatthe other cell-line(s) is a modulator of angiogenesis, as evidenced bythe difference in tubule formation potential. In some embodiments, thedifference between the tubule formation potential, for example asmeasured by the number of least one of the total area of the tubules,the total number of tubules, number of nodes, number of branch points,the number of tubes per node, or node area formed in the co-culture ofthe fluorescent cell-line of interest relative to a control is at leastabout 10%, such as at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 100%, at leastabout 150%, at least about 200%, at least about 250%, at least about300%, at least about 350%, at least about 400%, at least about 500%, orgreater than 500%. In some embodiments, the difference is astatistically significant difference. Thus, a cell-line can induce astatistically significant difference in the tubule formation potentialof a fluorescent cell-line of interest, such as one of the disclosedfluorescent cell-lines. Taking a combinatorial approach the impact ofmultiple different cell-lines either alone or in combination on thetubule formation potential of the fluorescent cell-line of interest canbe determined. In some examples, the presence of one or more additionalcell-lines decreases the tubule formation potential of the fluorescentcell-line of interest, for example as measured by the total area of thetubules, the total number of tubules, number of nodes, number of branchpoints, the number of tubes per node, or node area formed by thefluorescent cell-line of interest. These cell-lines would be identifiedas negative regulators of angiogenesis.

Utilizing fluorescent cell-lines, tubule formation assays can also beused to screen for a biological effect of a test agent, such as theeffect of potential modulators of angiogenesis, for example compoundsthat are structurally related to any one of Compounds 1-77 (TABLE 1),structurally related to the scaffolds presented in any one of TABLES2-9, derivatives or fragments of any of the compounds described herein,and so forth. In some embodiments, a fluorescent cell-line of interest(or multiple cell-lines of interest in a multiplex assay) is contactedwith an exogenous agent, such as a cell-line or test agent, and theimpact of the exogenous agent on tubule formation potential can bedetermined. Exemplary test agents are provided herein. For example usingthe difference between the total area of the tubules, the total numberof tubules, number of nodes, number of branch points, the number oftubes per node, and/or node area between a fluorescent cell-line ofinterest and a control are used to determine if an exogenous agent, suchas a test agent, impacts the ability of a fluorescent cell-line ofinterest to form tubules. A difference between the tubule formationpotential of a fluorescent cell-line of interest contacted with anexogenous agent and a control (such as a control culture exposed to theexogenous agent) indicates that the exogenous agent is a modulator ofangiogenesis. In some embodiments, the difference between the tubuleformation potential of the fluorescent cell-line contacted with anexogenous agent relative to a control is at least about 10%, such as atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%. Insome embodiments, the difference is a statistically significantdifference. Thus, an exogenous agent can induce a statisticallysignificant difference in the tubule formation potential of thefluorescent cell-line of interest contacted with the test agent, ascompared to the control, such as the fluorescent cell-line of interestnot contacted with the exogenous agent.

In one embodiment, the exogenous agent decreases the ability of afluorescent cell-line of interest to form tubules. A test agentexhibiting such an activity is identified as a potential inhibitor ofangiogenesis and would be of use in treating a disease or condition inwhich normal angiogenesis is increased, for example cancer. Such agentscould be used alone, or in combination with other agents (such as, butnot limited to a SM, peptide or antibody) known to inhibit angiogenesisby a similar or complementary mechanism (such as at a different step inthe angiogenesis pathway). In some embodiments, a decrease in the tubuleformation potential of the fluorescent cell-line of interest is at leastabout a 30%, at least about a 40%, at least about a 50%, at least abouta 60%, at least about a 70%, at least about a 80%, at least about a 90%,at least about a 100%, at least about a 150%, at least about a 200%, atleast about a 250%, at least about a 300%, at least about a 350%, atleast about a 400%, at least about a 500% decrease as compared tocontrol. In additional embodiments, the decrease is a statisticallysignificant decrease as compared to a control.

iii. Fluorescence-Based Migration Assay

Another assay that can be used is a cellular migration assay. Theseassays assess cellular migration in a controlled environment, such as adifferential migration of the cell-line, (or multiple cell-lines in amultiplex assay) as determined by fluorescent signals (such as theintensity of a fluorescent signal of a particular color, or at aparticular wavelength, such as the emission maxima of a particularfluorescent protein) in a location that is associated with migration toa particular location.

A cellular migration assay can be used to determine the ability of cellsto migrate up or down a chemical gradient. Migration “up” a chemicalgradient refers to migration from a region of lower chemicalconcentration of a chemical to a region of higher chemical concentration(for example migration toward a higher concentration of a chemicalattractant or away from a lower concentration of the chemicalattractant), while migration “down” a chemical gradient refers tomigration from a region of higher chemical concentration to a region oflower chemical concentration (for example migration away from a higherconcentration of a chemical repellent toward a lower concentration ofthe chemical repellent). Such migration is typically referred to aschemotaxis. Cells, such as fluorescent cell-lines, respond to chemicalsignals in their environment by the stimulation of concerted movementeither toward a chemical attractant or away from a chemical repellent.In mammalian cells, such as fluorescent cell-lines, typicalchemo-attractants include factors excreted by cells, for example factorsfound in serum, such as growth factors and the like.

Fluorescent cells (such as those described in U.S. application Ser. No.12/060,752 (published as US 2009/0088341 on Apr. 2, 2009)) can be usedin any cell migration assay format, such as the ChemoTx™ system(NeuroProbe, Rockville, Md.), transwell system or any other suitabledevice or system. In some examples, a cell migration assay is carriedout as follows: A culture of a fluorescent cell-line of interest isplaced into a first chamber of a cell migration apparatus, and anexogenous agent (such as a chemoattractant) is placed in a secondchamber that is adjacent to and in communication with the first chamberof the cell migration apparatus, so that cellular migration from thefirst chamber to the second chamber can be detected. The chambers may beseparated by a membrane or filter that permits passage of cells from onechamber to the other chamber. The membrane or filter is configured suchthat the passive diffusion of the cells across the membrane or filter isminimized. In one example, the first chamber is the upper chamber of theapparatus and the second chamber is the lower chamber of the apparatus.In some examples the upper chamber is omitted and the cells are placeddirectly on a membrane or filter in communication with the lowerchamber. The ability of a fluorescent cell-line such as the fluorescentcell-lines used in the assays described herein to be stimulated tomigrate can be determined. Typical migration assays have “unknown” sites(with cell suspension above the filter and a solution containing thechemotactic factor below it) and “negative control” sites (with cellsuspension above the filter and suspension media, but no chemotacticfactor, below). Random migration of unstimulated cells will account forsome of the cells that pass through the filter. Migrated cells at thenegative control sites show the extent of unstimulated random migration,which can then be differentiated from chemotactic migration, orchemotaxis. Cells that stably express a fluorescent protein, such as thedisclosed fluorescent cells can be read in a microplate with afluorescence microplate reader. Thus, the number of fluorescent cellspresent in either the upper chamber, lower chamber, or both chambers canbe determined, for example as a function of time.

Migration assays can be used to determine if an exogenous agent, such asa test agent, affects or differentially affects the migration of one ormore of the fluorescent cell-line of interest. A fluorescent cell-lineof interest can be contacted with exogenous agent and the impact of theexogenous agent on the migration of the fluorescent cell-line ofinterest can be determined. For example, a difference between the numberof cells that migrate between a fluorescent cell-line of interestcontacted with an exogenous agent and a control indicates that theexogenous agent, such as a test agent, cell-line, growth factor, etc.,is a modulator of cellular migration. In other embodiments, differencesin migration among different cell-lines in the migration assay providean indication of differential migration of the different cell-lines inresponse to the exogenous agent. In some embodiments, the differencebetween the number of cells that migrate of the fluorescent cell-linecontacted with an exogenous agent relative to a control, (for example asmeasured by the fluorescence intensity of a fluorescent protein stablyand constitutively expressed by the cells) is at least about 10%, suchas at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%. Insome embodiments, the difference is a statistically significantdifference. Thus, an exogenous agent can induce a statisticallysignificant difference in the migration of a fluorescent cell-line ofinterest contacted with the exogenous agent, as compared to the control,such as the fluorescent cell-line of interest not contacted with theexogenous agent or a different cell-line that has been mixed with thecell-line of interest.

In one embodiment, the exogenous agent, such as a test agent, decreasesthe ability of a fluorescent cell-line of interest to migrate. A testagent with such an activity is identified as a potential inhibitor ofangiogenesis and would be of use in treating a disease or condition inwhich normal angiogenesis is increased, for example cancer. In someembodiments, a decrease in migration of the fluorescent cell-line ofinterest is at least about a 30%, at least about a 40%, at least about a50%, at least about a 60%, at least about a 70%, at least about a 80%,at least about a 90%, at least about a 100%, at least about a 150%, atleast about a 200%, at least about a 250%, at least about a 300%, atleast about a 350%, at least about a 400%, at least about a 500%decrease as compared to control. In additional embodiments, the decreaseis a statistically significant decrease as compared to a control.

iv. Fluorescence-Based Cell Viability Assay

Another example of an assay is a cell viability assay. Such assays arebased on the release of fluorescent protein from the cytoplasm offluorescent cell-lines that constitutively express fluorescent proteinthat occurs when the integrity of the cell membrane of the cells iscompromised, for example when the cell dies, such as when the cell isexposed to a cytotoxic agent, such as a test agent that is cytotoxic tothe cell. Upon exposure to a cytotoxic agent the fluorescent protein isliberated to the culture media and it can be measured, for example usinga fluorimeter. The greater the amount of fluorescent protein liberatedfrom the cells present in the culture, the greater the intensity of thefluorescence present in the media. The measured fluorescence in themedia corresponds to number of dead cells.

In some embodiments, the cell viability assay is used to determine if anexogenous agent, such as a test agent, is cytotoxic to one or more ofthe fluorescent cell-lines of interest, such as one or more of thefluorescent cell-lines disclosed herein. A fluorescent cell-line ofinterest can be contacted with exogenous agent and the impact of theexogenous agent on the death of the fluorescent cell-line of interestcan be determined. For example, an increase in the relative florescencepresent in the media of between a fluorescent cell-line of interestcontacted with an exogenous agent and a control indicates that theexogenous agent, such as a test agent, cell-line, growth factor, etc.,is cytotoxic to the cell-line of interest. In other embodiments,differential cytotoxicity of an exogenous agent to different cell-linesin the cell viability assay provides an indication that a specificexogenous agent is preferentially cytotoxic to one cell-line but notother cell-lines present in the culture. Such information is useful forscreening agents that are preferentially or differentially cytotoxic toa specific cell-type, for example to the exclusion of other cell types.For example, in a mixed cell population a test agent could be screenedto determine if it was cytotoxic (for example differentially cytotoxic)to diseased cells (such as tumor cells) present in the mixed cellpopulation, but not normal cells present in the mixed cell population.

In some embodiments, the difference between the fluorescence of themedia of a fluorescent cell-line contacted with an exogenous agentrelative to a control, (for example as measured by the fluorescenceintensity of a fluorescent protein liberated from the cell-line into themedia) is at least about 10%, such as at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about100%, at least about 150%, at least about 200%, at least about 250%, atleast about 300%, at least about 350%, at least about 400%, at leastabout 500%, or greater than 500%. In some embodiments, the difference isa statistically significant difference. Thus, an exogenous agent, suchas a test agent, can induce a statistically significant difference inthe number of cells that die as a the migration of a fluorescentcell-line of interest contacted with the exogenous agent, as compared tothe control, such as the fluorescent cell-line of interest not contactedwith the exogenous agent or a different cell-line that has been mixedwith the cell-line of interest.

v. Additional Angiogenesis Assays

The following descriptions provide additional examples of angiogenicassays, which may be useful in measurements of the angiogenic (orantiangiogenic) activity of a test compound, such as a compoundstructurally related to one of Compounds 1-77 (TABLE 1), or a scaffoldshown in any of TABLES 2-9, and derivatives thereof. In particularexamples, these assays can also be used to measure the antiangiogenicactivity of combinations of at least two SMs from Compounds 1-77 orcombinations of at least one SM from Compounds 1-77 and an additionalknown or potential angiogenesis inhibitor. One of ordinary skill in theart will recognize that other angiogenic assays also can be used.

Corneal Pocket Assay:

This is the “gold standard” method for following the effect of definedsubstances to promote neovascularization of the normally avascularcornea. This assay has the advantage that new blood vessels are easilydetected and essentially must be newly formed blood vessels in thenormally avascular cornea. Agents to be tested for angiogenic oranti-angiogenic activity are immobilized in a slow release form in aninert hydron pellet of approximately 1-2 μl volume. That pellet isimplanted into the corneal epithelium of an anesthetized C57BL mouse (ora rabbit) in a pocket created by micro-dissection. Over a five to sevenday period angiogenic factors stimulate the ingrowth of vessels from theadjacent vascularized corneal limbus. A photographic record is createdby slit lamp photography. The appearance, density and extent of thesevessels are evaluated and scored. In some cases, the time course of theprogression is followed in anesthetized animals, prior to sacrifice.Vessels are evaluated for length, density and the radial surface of thelimbus from which they emanate (expressed as clock-faced hours).

Intradermal Sponge Angiogenesis Assay:

Inert biopolymer sponges impregnated with defined amounts of testreagents are implanted subcutaneously through a transdermal incision,into a pocket created in the subcutaneous tissue. Sponges are thenremoved following a defined periods ranging from five to fifteen daysand the new vessel formation quantitated by a number of biochemical andhistomorphometric parameters. Portions of a sponge can be extracted andanalyzed by Western blot for endothelial restricted gene product such asVE cadherin, FLK-1 receptors, and others. Frozen section portions ofthat same sample are evaluated by immunohistochemistry for similarantigens to confirm that expression levels reflect endothelial cellproteins contained within new vessels that have invaded the sponge. Inconjunction with the mouse corneal pocket assay, systemic administrationof putative angiogenesis inhibitors by intraperitoneal or intravenousroutes permits evaluation and comparison of the local effects of thoseinhibitors on angiogenic stimuli in different microvascular beds.

Chick Chorioallantoic Membrane (CAM) Assay:

Another assay involves the use of chicken chorioallantoic membrane (theCAM assay; see Wilting et al., Anat. Embryol. 183: 259, 1991). The CAMassay permits the quantitation of angiogenesis and anti-angiogenesis inthe chick embryo chorioallantoic membrane (CAM). Briefly, chicken eggsare windowed on day two or three of incubation and the windows aresealed with tape, wax, glass slides, or PARAFILM® wrapper. On day eightof incubation, the windows are opened, and small sponges or pieces ofgelatin are placed on top of the growing CAM.

After implantation, the sponges are treated with at least one stimulatororinhibitor (for example, any of Compounds 1-77) of blood vesselformation. Blood vessels growing vertically into the sponge and at theboundary between sponge and surrounding CAM mesenchyme are counted by amorphometric method on day twelve. Factors that increase the number ofblood vessels growing into the sponge are considered angiogenic, whereasfactors that inhibit blood vessel growth into the sponge are consideredantiangiogenic. Quantification of the number of new vessels yields ameasure of angiogenicity. Thus, this technique facilitates thecharacterization of agonists or antagonists of angiogenesis. (For moreinformation, see Ribatti et al., J. Vasc. Res. 1997, 34:455-463).

Directed In Vivo Angiogenesis Assay (DIVAA):

Yet another angiogenesis assay is termed a Directed in vivo AngiogenesisAssay (DIVAA; Guedez et al., American Journal of Pathology162(5):1431-9, 2003). Silicone tubes (0.15 mm outside diameter, New AgeIndustries, Southampton, Pa.) are cut to 1 cm in length, and one end ofeach tube is closed with liquid silicone and dried for 24 hours, thenautoclaved. A dilution of test substances is prepared in matrigel insterile cold Eppendorf tubes. Tubes are filled with a Hamilton syringe.Nude mice are anesthetized, and a pocket is made in the dorsal skin ofeach animal. The tubes are then implanted with the open end first andthe wounds are sealed.

After nine to eleven days, the tail veins are injected with FITC-dextranto visualize the blood vessels, and the dye is allowed to distributethroughout the vasculature for about 20 minutes. Mice are theneuthanized with CO₂ and the skin pockets are removed.

Skin is then dissected, keeping the vessels near the mouth of the tube.The matrigel is then displaced from the tube, incubated at 37° C. in thepresence of dispase, then vortexed, centrifuged, and matrigel aliquotsare transferred into 96-well plates for fluorescent emission.Fluorescence is read in a fluorimeter.

VII. Pharmaceutical Compositions and Modes of Administration

The compounds described herein (such as Compounds 1-77), and derivativesthereof, are particularly useful for inhibiting or reducing angiogenesisin a subject, such as a subject suffering from a disease or conditionaccompanied by deregulated angiogenesis. The methods of inhibiting orreducing angiogenesis include administering to a subject atherapeutically effective amount of at least one agent identified as onethat inhibits or reduces angiogenesis (e.g., any of Compounds 1-77, asdescribed herein). Thus in some embodiments, the pharmaceuticalcomposition containing a bioactive compound that decreases angiogenesisis administered to a subject, such as a subject with cancer or anotherdisease or condition which would be treated by reducing angiogenesis. Insome embodiments, the subject is a human subject. It is alsocontemplated that the pharmaceutical compositions containing at leastone bioactive compound that decreases angiogenesis can be administeredwith known conventional treatments, for instance treatments for cancer,such as in conjunction with a therapeutically effective amountchemotherapeutic agent.

In particular embodiments, the pharmaceutical composition comprises atleast one of 2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride(NSC 150117), deoxybouvardin (NSC 259969), (2,5-dioxopyrrol-1-yl)methylpropanoate (NSC 19630), 1-benzylsulfonyl-2,4-dinitrobenzene (NSC122657), maytansinol isobutyrate (NSC 292222), chloroplatinum(1+);2-(4-methylpiperidin-1-yl)ethanethiolate; dehydrate (NSC 292596), or apharmaceutically acceptable salt thereof. The composition can alsocomprise any combination of two, three, four, five, or six of thesecompounds. In other particular embodiments, any of the describedcompositions further comprise [4-[(4-arsonophenyl)methyl]phenyl]arsonicacid (NSC 48300), or a pharmaceutically-acceptable salt thereof. Thecombinations of the compounds can be determined based in part on thedifferential effect of each of the SMs in the combination on expressionof angiogenesis genes, as described in greater detail in Example 5.

Therapeutic compound(s) can be administered directly to a subject forexample a human subject. Administration is by any of the routes normallyused for introducing a compound into ultimate contact with the tissue tobe treated. The compounds are administered in any suitable manner,optionally with pharmaceutically acceptable carrier(s). Suitable methodsof administering therapeutic compounds are available and well known tothose of skill in the art, and although more than one route can be usedto administer a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

When the antiangiogenic compound is to be used as a pharmaceutical, itis placed in a form suitable for therapeutic administration. The testagent (antiangiogenic compound) may, for example, be included in apharmaceutically acceptable carrier such as excipients and additives orauxiliaries, and administered to a subject. Frequently used carriers orauxiliaries include magnesium carbonate, titanium dioxide, lactose,mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, nontoxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: MackPublishing Co., 1405-1412, 1461-1487, 1975, and The National FormularyXIV, 14th ed., Washington: American Pharmaceutical Association, 1975).The pH and exact concentration of the various components of thepharmaceutical composition are adjusted according to routine skills inthe art. See Goodman and Gilman The Pharmacological Basis forTherapeutics, 7th ed.

The pharmaceutical compositions are in general administered topically,intravenously, orally or parenterally or as implants. Suitable solid orliquid pharmaceutical preparation forms are, for example, granules,powders, tablets, coated tablets, (micro)capsules, suppositories,syrups, emulsions, suspensions, creams, aerosols, drops or injectablesolution in ampoule form and also preparations with protracted releaseof active compounds, in whose preparation excipients and additivesand/or auxiliaries such as disintegrants, binders, coating agents,swelling agents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science, 249:1527-1533,1990, which is incorporated herein by reference.

For treatment of a patient, depending on activity of the compound,manner of administration, nature and severity of the disorder, age andbody weight of the patient, different daily doses are necessary. Undercertain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units, and also by multiple administrations ofsubdivided doses at specific intervals.

A therapeutically effective dose is the quantity of a compound accordingto the disclosure necessary to prevent, to cure or at least partiallyameliorate the symptoms of a disease and its complications. Amountseffective for this use will, of course, depend on the severity of thedisease and the weight and general state of the patient. Typically,dosages used in vitro may provide useful guidance in the amounts usefulfor in situ administration of the pharmaceutical composition, and animalmodels may be used to determine effective dosages for treatment ofparticular disorders. Various considerations are described, e.g., inGilman et al., eds., Goodman and Gilman: the Pharmacological Bases ofTherapeutics, 8th ed., Pergamon Press, 1990; and Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa.,1990. Effectiveness of the dosage can be monitored by any method.

The antiangiogenic compounds described herein may be formulated in avariety of ways depending on the location and type of disease to betreated or prevented. Pharmaceutical compositions are thus provided forboth local use at or near an affected area and for systemic use (inwhich the agent is administered in a manner that is widely disseminatedvia the cardiovascular system). This disclosure includes within itsscope pharmaceutical compositions including at least one antiangiogeniccompound, formulated for use in human or veterinary medicine.

Pharmaceutical compositions that include at least one antiangiogeniccompound as described herein as an active ingredient, or that includeboth an antiangiogenic compound and an additional anti-angiogenic agent,may be formulated with an appropriate solid or liquid carrier, dependingupon the particular mode of administration chosen. Additional activeingredients include, for example, anti-angiogenic agents, such asinhibitors of bFGF or VEGF.

A suitable administration format may best be determined by a medicalpractitioner for each subject individually. Various pharmaceuticallyacceptable carriers and their formulation are described in standardformulation treatises, for example, Remington's Pharmaceutical Sciencesby E. W. Martin. See also Wang and Hanson, J. Parenteral Sci. Technol.,Technical Report No. 10, Supp. 42: 2S, 1988.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, inhalational, topical, ophthalmic, peritoneal, andoral formulations can be employed. Inhalational preparations can includeaerosols, particulates, and the like. In general, the goal for particlesize for inhalation is about 1 μm or less in order that thepharmaceutical reach the alveolar region of the lung for absorption.Oral formulations may be liquid (for example, syrups, solutions, orsuspensions), or solid (for example, powders, pills, tablets, orcapsules). For solid compositions, conventional non-toxic solid carrierscan include pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. Actual methods of preparing such dosage forms areknown, or will be apparent, to those of ordinary skill in the art.

The compositions or pharmaceutical compositions can be administered byany route, including parenteral administration, for example,intravenous, intramuscular, intraperitoneal, intrasternal, orintra-articular injection or infusion, or by sublingual, oral, topical,intra-nasal, ophthalmic, or transmucosal administration, or by pulmonaryinhalation. When anti-angiogenic compounds are provided as parenteralcompositions, for example, for injection or infusion, they are generallysuspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 3.0 to about 8.0, preferably at a pH of about3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffersinclude sodium citrate-citric acid and sodium phosphate-phosphoric acid,and sodium acetate/acetic acid buffers. A form of repository or “depot”slow release preparation may be used so that therapeutically effectiveamounts of the preparation are delivered into the bloodstream over manyhours or days following transdermal injection or delivery.

Antiangiogenic compounds are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseformulations include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, forexample, films, or mirocapsules), suitable hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, andsparingly soluble derivatives (such as, for example, a sparingly solublesalt). Sustained-release antiangiogenic compounds may be administered byintravascular, intravenous, intra-arterial, intramuscular, subcutaneous,intra-pericardial, or intra-coronary injection. Administration can alsobe oral, rectal, parenteral, intracisternal, intravaginal,intraperitoneal, topical (as by powders, ointments, gels, drops ortransdermal patch), buccal, or as an oral or nasal spray.

Preparations for administration can be suitably formulated to givecontrolled release of antiangiogenic compounds. For example, thepharmaceutical compositions may be in the form of particles comprising abiodegradable polymer and/or a polysaccharide jellifying and/orbioadhesive polymer, an amphiphilic polymer, an agent modifying theinterface properties of the particles and a pharmacologically activesubstance. These compositions exhibit certain biocompatibility featuresthat allow a controlled release of the active substance. See, forexample, U.S. Pat. No. 5,700,486.

In some embodiments, antiangiogenic compounds are delivered by way of apump (see Sefton, CRC Crit. Ref. Biomed. Eng. 14:201, 1987; Buchwald etal., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574,1989) or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The keyfactor in selecting an appropriate dose is the result obtained, asmeasured by increases or decreases in angiogenesis, or by other criteriafor measuring control or prevention of disease, as are deemedappropriate by the practitioner. Other controlled release systems arediscussed in the review by Langer (Science 249:1527-1533, 1990).

In another aspect of the disclosure, antiangiogenic compounds aredelivered by way of an implanted pump, described, for example, in U.S.Pat. No. 6,436,091; U.S. Pat. No. 5,939,380; and U.S. Pat. No.5,993,414. Implantable drug infusion devices are used to providesubjects with a constant and long term dosage or infusion of a drug orany other therapeutic agent. Essentially, such device may be categorizedas either active or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the drug into the patient's system. Anexample of such an active drug infusion device currently available isthe Medtronic SynchroMed™ programmable pump. Such pumps typicallyinclude a drug reservoir, a peristaltic pump to pump the drug out fromthe reservoir, and a catheter port to transport the pumped out drug fromthe reservoir via the pump to a patient's anatomy. Such devices alsotypically include a battery to power the pump, as well as an electronicmodule to control the flow rate of the pump. The Medtronic SynchroMed™pump further includes an antenna to permit the remote programming of thepump.

Passive drug infusion devices, in contrast, do not feature a pump, butrather rely upon a pressurized drug reservoir to deliver the drug. Thus,such devices tend to be both smaller as well as cheaper as compared toactive devices. An example of such a device includes the MedtronicIsoMed™. This device delivers the drug into the patient through theforce provided by a pressurized reservoir applied across a flow controlunit.

The implanted pump can be completely implanted under the skin of asubject, thereby negating the need for a percutaneous catheter. Theseimplanted pumps can provide the patient with antiangiogenic compounds ata constant or a programmed delivery rate. Constant rate or programmablerate pumps are based on either phase-change or peristaltic technology.When a constant, unchanging delivery rate is required, a constant-ratepump is well suited for long-term implanted drug delivery. If changes tothe infusion rate are expected, a programmable pump may be used in placeof the constant rate pump system. Osmotic pumps may be much smaller thanother constant rate or programmable pumps, because their infusion ratecan be very low. An example of such a pump is described listed in U.S.Pat. No. 5,728,396.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). The tablets can be coated bymethods well known in the art. Liquid preparations for oraladministration can take the form of, for example, solutions, syrups orsuspensions, or they can be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations can be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (for example, sorbitolsyrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (for example, lecithin or acacia); non-aqueous vehicles (forexample, almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate.

For administration by inhalation, the compounds for use according to thepresent disclosure are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

For topical administration, the compounds for use are, for example,mixed with ethanol, methanol, propylene glycol, or dimethyl sulfoxide,which act as a vehicle to facilitate uniform distribution of thecompound to a target area of the subject's body, such as a wound ordecubitus ulcer.

Pharmaceutical compositions that comprise an antiangiogenic compound asdescribed herein as an active ingredient will normally be formulatedwith an appropriate solid or liquid carrier, depending upon theparticular mode of administration chosen. The pharmaceuticallyacceptable carriers and excipients useful in this disclosure areconventional. For instance, parenteral formulations usually compriseinjectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like.Excipients that can be included are, for instance, proteins, such ashuman serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in the art.

For example, for parenteral administration, antiangiogenic compounds canbe formulated generally by mixing them at the desired degree of purity,in a unit dosage injectable form (solution, suspension, or emulsion),with a pharmaceutically acceptable carrier, for instance, one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. Apharmaceutically acceptable carrier is a non-toxic solid, semisolid orliquid filler, diluent, encapsulating material or formulation auxiliaryof any type.

Generally, the formulations are prepared by contacting theantiangiogenic compounds each uniformly and intimately with liquidcarriers or finely divided solid carriers or both. Then, if necessary,the product is shaped into the desired formulation. Optionally, thecarrier is a parenteral carrier, and in some embodiments it is asolution that is isotonic with the blood of the recipient. Examples ofsuch carrier vehicles include water, saline, Ringer's solution, anddextrose solution. Non-aqueous vehicles such as fixed oils and ethyloleate are also useful herein, as well as liposomes.

The pharmaceutical compositions that comprise an antiangiogeniccompound, in some embodiments, will be formulated in unit dosage form,suitable for individual administration of precise dosages. The amount ofactive compound(s) administered will be dependent on the subject beingtreated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

The therapeutically effective amount of antiangiogenic compound will bedependent on the specific compound utilized, the subject being treated,the severity and type of the affliction, and the manner ofadministration.

VII. Therapeutic Uses

Methods are disclosed herein for inhibiting angiogenesis in a subject(or in an area in a subject) who has or is at risk for developing, forinstance, a tumor (whether malignant or benign), retinopathy, psoriasis,endometriosis, arthritis, or any other disease for which it would bebeneficial to inhibit angiogenesis. The methods include introducing atherapeutically effective amount of an antiangiogenic compound (e.g.,one of Compounds 1-77) to the area (or systemically), thereby inhibitingangiogenesis in the subject.

In one embodiment, the angiogenesis inhibitory compound is administeredlocally. For subjects with a tumor, administration may be, for example,by intra-arterial injection to the tumor's arterial supply, or by directinjection into the tumor. Other routes of administration will bedetermined by the tumor location. Ovarian tumors are, for example,treated by intraperitoneal washing with the inhibitor. A brain tumor is,for example, treated by intra-arterial or intrathecal injection, byintranasal administration, by direct injection of affected brain areas,or by intravenous or intra-arterial injection following osmoticdisruption of the blood brain barrier (see, for example, U.S. Pat. No.5,124,146). Lung cancer is treated, for example, by direct injection ofthe tumor, by inhalation, or infusion into the lobar circulation of anaffected lobe of the lung. Efficacy of the treatment is determined, forexample, by monitoring tumor burden, or is indicated, for example, by alessening of symptoms, such as pain.

For subjects with retinopathy, administration is, for example, byintra-ocular injection (for example, into the posterior chamber of theeye), or by topical ophthalmic administration. Alternatively, the agentmay be administered intravascularly, for example into the vascularsupply for the retinal artery. Efficacy of the treatment is determined,for example, by an improvement in vision, by a stabilization of vision,by a lack of new blood vessel formation in the retina, or by failure ofthe disease to progress.

For subjects with psoriasis, administration is, for example, bysubcutaneous or intravenous injection, or by topical application.Efficacy of the treatment is determined, for example, by an abatement ofpsoriasis symptoms. For subjects with arthritis, administration is, forexample, by intra-articular injection. Efficacy of the treatment ismonitored, for example, by detecting an improvement in mobility, or alessening of joint pain. For subjects with endometriosis, administrationis, for example, by direct injection of the endometrial growths, or byintraperitoneal washing with the antiangiogenic compound. Efficacy ofthe treatment is shown, for example, by an improvement in mobility, or alessening of pelvic pain.

Administration of the angiogenesis inhibitor may begin whenever asubject has developed, or is at risk for developing a tumor,retinopathy, psoriasis, or endometriosis, or when symptoms ofinappropriate neovascularization are present.

Also disclosed are methods for treating undesirable angiogenesis andangiogenesis dependent or associated diseases, in a subject. The methodincludes administering one or more of the presently described compounds,or a combination of one or more of the compounds and one or more otherpharmaceutical agents, to the subject in a pharmaceutically compatiblecarrier. The administration is made in an amount effective to inhibitthe development or progression of angiogenesis and diseases associatedwith the same. Although the treatment can be used prophylactically inany patient in a demographic group at significant risk for suchdiseases, subjects can also be selected using more specific criteria,such as a definitive diagnosis of the condition.

The vehicle in which the drug is delivered can include pharmaceuticallyacceptable compositions of the drugs, using methods well known to thosewith skill in the art. Any of the common carriers, such as sterilesaline or glucose solution, can be utilized with the drugs disclosedherein. Routes of administration include but are not limited to oral andparenteral routes, such as intravenous (iv), intraperitoneal (ip),rectal, topical, ophthalmic, nasal, and transdermal.

The drug may be administered in a suitable manner now known or laterdeveloped, e.g., orally or intravenously, in any conventional medium.For example, intravenous injection may be by an aqueous saline medium.The medium may also contain conventional pharmaceutical adjunctmaterials such as, for example, pharmaceutically acceptable salts toadjust the osmotic pressure, lipid carriers such as cyclodextrins,proteins such as serum albumin, hydrophilic agents such as methylcellulose, detergents, buffers, preservatives and the like. A morecomplete explanation of parenteral pharmaceutical carriers can be foundin Remington: The Science and Practice of Pharmacy (19^(th) Edition,1995) in chapter 95.

Examples of other pharmaceutical compositions can be prepared withconventional pharmaceutically acceptable carriers, adjuvants andcounterions as would be known to those of skill in the art. Thecompositions are preferably in the form of a unit dose in solid,semi-solid and liquid dosage forms such as tablets, pills, powders,liquid solutions or suspensions.

The compounds illustrated herein are ideally administered as soon aspossible after unwanted angiogenesis is detected. For example, onceunwanted angiogenesis has been confirmed or the presence of a tumor hasbeen identified, a therapeutically effective amount of the drug isadministered. The compound(s) can be administered in a single dose, orin multiple doses, for example daily, weekly, every two weeks, ormonthly during a course of treatment.

Therapeutically effective doses of the presently described compounds canbe determined by one of skill in the art, with a goal of achieving adesired level of antiangiogenesis as illustrated in the foregoingexamples. In one embodiment, an antiangiogenic effective amount is anamount sufficient to achieve a statistically significant inhibition ofangiogenesis compared to a control. Angiogenesis can be readily assessedusing an assay, e.g., any of the assays described herein. Alternatively,angiogenesis can be determined in another assay or by direct or indirectsigns of angiogenesis in a patient.

The relative toxicities of the compounds make it possible to administerin various dosage ranges. An example of such a dosage range is fromabout 0.5 to about 50 mg/kg body weight orally in single or divideddoses. Another example of a dosage range is from about 1.0 to about 25mg/kg body weight orally in single or divided doses. For oraladministration, the compositions are, for example, provided in the formof a tablet containing from about 25 to about 500 mg of the activeingredient, particularly 100 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the specific compound, the extent of existingangiogenic activity, the age, body weight, general health, sex, diet,mode and time of administration, rate of excretion, drug combination,and severity of the condition of the host undergoing therapy.

The pharmaceutical compositions containing at least one of theantiangiogenic SMs described herein can be used in the treatment of avariety of diseases mediated by angiogenesis. Examples of suchangiogenesis-dependent diseases include all types of cancer, ocularneovascular disease, tumor formation and metastasis in tumors such asmyeloma, rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, osteosarcoma, colon, prostate, head and neck, breast,bladder, liver, pancreatic, lung, CNS, and blood-born tumors such asleukemia, also diseases such as hemangioma, ulcerative colitis, Crohn'sdisease, diabetic retinopathy, macular degeneration, sickle cell anemia,sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, veinocclusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eale's disease,Bechet's disease, infections causing a retinitis or choroiditis,presumed ocular histoplasmosis, Best's disease, myopia, optic pits,Stargart's disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, trauma and post-lasercomplications. Other diseases include, but are not limited to, diseasesassociated with rubeosis (neovasculariation of the angle) and diseasescaused by the abnormal proliferation of fibrovascular or fibrous tissueincluding all forms of proliferative vitreoretinopathy.

Also disclosed herein are combinations of one or more of the presentlydescribed compounds with one or more of various other angiogenesisinhibitor compounds. For example, the presently described compounds maybe administered in combination with effective doses of otherantiangiogenic agents. The term “administration in combination” refersto both concurrent and sequential (in either order) administration ofthe active agents. Non-limiting examples of antiangiogenic agents thatcan be used in combination with the compounds indentified herein areTNP-470, carbonic anhydrase inhibitors, endostatin, angiostatin,2-methoxyestradiol, IMiD (Immune-modulating inhibitor drug) CC5013,matrix metalloproteinase inhibitors, and COL-3, as well as bFGF or VEGFinhibitor, such as SU5416, which is a specific VEGF-R antagonist, andSU6668 which blocks the receptors for VEGF, bFGF, and PDGF (see, forexample, Liu et al., Seminars in Oncology 29 (Suppl 11): 96-103, 2002;Shepherd et al., Lung Cancer 34:S81-S89, 2001). In addition, thepresently described compounds may be used in combination with otherforms of cancer therapy (e.g., chemotherapy, radiation therapy, hormonaltherapy) or other therapies known for use with angiogenesis-relateddisorders and diseases.

ADDITIONAL REFERENCES

-   1. Blower et al., Comparison of methods for sequential screening of    large compound sets. Comb Chem High Throughput Screen 9(2):115-22,    2006-   2. Blower et al., Systematic analysis of large screening sets in    drug discovery. Curr Drug Discov Technol 1(1):37-47, 2004-   3. Cross et al., Finding discriminating structural features by    reassembling common building blocks. J Med Chem 46(22):4770-5, 2003-   4. Gagarin et al., Using clustering techniques to improve hit    selection in high-throughput screening. J Biomol Screen    11(8):903-14, 2006-   5. Hopkins, Network pharmacology: the next paradigm in drug    discovery. Nat Chem Biol 4(11):682-90, 2008-   6. Makarenkov et al., HTS-Corrector: software for the statistical    analysis and correction of experimental high-throughput screening    data. Bioinformatics 22(11):1408-9, 2006-   7. Makarenkov et al., An efficient method for the detection and    elimination of systematic error in high-throughput screening.    Bioinformatics 23(13):1648-57, 2007-   8. Malo et al., Statistical practice in high-throughput screening    data analysis. Nat Biotechnol 24(2):167-75, 2006-   9. Yang et al., Building predictive models for protein tyrosine    phosphatase 1B inhibitors based on discriminating structural    features by reassembling medicinal chemistry building blocks. J Med    Chem 47(24):5984-94, 2004-   10. Zhang et al., A Simple Statistical Parameter for Use in    Evaluation and Validation of High Throughput Screening Assays. J    Biomol Screen 4(2):67-73, 1999

EXAMPLES Example 1 Antiangiogenic Small Molecule Signatures

This example describes the identification and initial characterizationof antiangiogenic small molecules using cell-based HTS methodology.

The present disclosure relates to the discovery of a new set ofantiangiogenic small molecules. The antiangiogenic small molecules wereidentified through a combination of cell-based high throughput screening(HTS) together with chemo-informatic tools. This approach differs fromthe one taken by previous studies and pharmaceutical companies, in thatthe HTS is not targeted to a single subcellular molecule but insteadtargets a whole cellular process. In particular, two cell based assayshave been developed which represent the two most important steps inangiogenesis: endothelial cell growth and tube formation.

Using the cell-based HTS screen, a new set of antiangiogenic smallmolecules have been discovered. Structure-activity-relationship (SAR)studies have shown that most of these new bioactive SMs are not relatedto known antiangiogenic SMs (FDA marketed; SMs currently in clinicaltrials; SMs annotated as antiangiogenic in chemical databases such asLeadScope, DrugBank, PubChem, etc.).

Overview of High Throughput Screen

The small molecule library screened was the NCI Diversity Set I(available on-line atdtp.nci.nih.gov/branches/dscb/diversity_explanation.html). The librarywas obtained from DTP/NCI (available on-line at dtp.nci.nih.gov/). Thislibrary contains 1974 small molecules (SMs) which are representativemembers of the same number of structural families each containing avariable number of members. The SMs included in the NCI Diversity Set Iwere selected to summarize the structural diversity found in a libraryof approximately 72,000 SMs. The library was obtained in 96-well platesand plate-to-plate DMSO dilutions were prepared at a stock concentrationof 200 μM.

Two different cell-based HTS assays were developed which mimic the twomain steps of the angiogenesis process: (1) a growth assay and (2) atube formation assay (see below for more detail on assay protocols). Theprimary goal of the two HTSs was to find SMs which block either growthor tube formation of endothelial cells. These two assays comprised theprimary screening which defined a first group of “bioactive compounds”which were then evaluated with secondary screening. See FIG. 1.

The secondary screening included two steps: (1) a cytotoxicity assay(described below) which was designed to discriminate cytotoxic compoundsand (2) calculation of IC50 for every of the compounds identified in theprimary screening. This secondary screening was also designed toeliminate putative false positives found in the primary screening and toevaluate the potency of the bioactive compounds. See FIG. 1.

The set of biologically active compounds obtained after the secondaryscreening was then used in a number of chemoinformatic studies as wellas in in vitro studies in order to partially characterize theirmechanism of action as well as compare them with existing antiangiogenicsmall molecules currently approved by the FDA, or in development bypharmaceutical companies.

Based on the compiled results of the HTS and other in vitro assays, asmall set of compounds was chosen for in vivo studies. These studiesincluded primarily xenograft experiments (See Example 2) and weredesigned to confirm the antiangiogenic activity of the compounds ofinterest, as well as test innovative anti-tumor/anti-angiogenicdrug-combination regimens.

Experimental Design for HTS

FIG. 2 illustrates the basic experimental design for all HTS assays; thesame scheme was applied to both growth and tube formation assays. Allassays were performed in clear bottom black 96-well plates. The plateswere always arranged to include a column for negative controls (column1), a column for positive controls (column 12), and 10 columns forevaluation of 80 compounds. In order to increase stringency and avoidfalse positives in the HTS assays, all compounds were tested at a lowfinal concentration of 1 μM.

Both HTS assays (growth and tube formation) were based on the use offluorescent reporter cell lines, essentially as described in U.S.application Ser. No. 12/060,752 (published as US 2009/0088341 on Apr. 2,2009; incorporated herein by reference in its entirety). In summary, thecell lines porcine aortic endothelial cell (PAE), BEC (a humanmicrovascular endothelial cell line), HMEC-1 (human microvascularendothelial cell line), A549 (human adenocarcinoma from the lung) andMCF7 (breast cancer cell line), among others, were stably transfectedwith different fluorescent proteins (green fluorescent protein -GFP-,yellow fluorescent protein -YFP-, red fluorescent protein -RFP-, andblue fluorescent protein -BFP-). Details on the production of these celllines are described in U.S. application Ser. No. 12/060,752, whichpublished as US 2009/0088341 on Apr. 2, 2009.

Growth HTS:

Previously, we demonstrated that there is a linear correlation betweenthe fluorescence emission of the reporter cell lines and their number inculture. In summary, 1,000 cells/well were seeded in the 96-well platesdescribed above. Cells in columns 2-12 were seeded in 10% FBS and cellsin column 1 (negative control) were seeded in 0% FBS Small molecules (80per plate) were added in columns 2-11 at a final concentration of 1 μM(both positive and negative controls were exposed to the same percentageof DMSO in wells containing test compounds). The fluorescence emitted byevery well was measured spectrophotometrically (Infinite M200, TECAN®,Mannedorf, Switzerland) every 24 hours for 5-7 days. The assay qualitywas evaluated y calculating the Z′ score (Zhang et al., J. Biomol.Screen 4(2):67-73, 1999) for every plate (only plates with Z′>0.5 wereconsidered).

Tube Formation HTS:

In summary, 20,000 cells/well were seeded on top of 50 μl of pre-gelledGELTREX® gel matrix (Invitrogen, Carlsbad, Calif.). All cells wereseeded in 10% FBS. Cells in column 1 (negative control) were exposed to25 μM of suramine (an antiangiogenic factor known to be an inhibitor ofendothelial tube formation). Small molecules (80 per plate) were addedto columns 2-11 at a final concentration of 1 μM (both positive andnegative controls were exposed to the same percentage of DMSO in wellscontaining test compounds). Plates were incubated for 5-7 hours andautomatically imaged with the help of an epifluorescence microscope(Axiovert® 200M, Zeiss) equipped with a motorized stage and AxioVision®(Zeiss) software. Images were analyzed with the AngioApplication™software (see below).

Hit Detection:

Data obtained from both the growth and tube formation HTSs wereprocessed with the HTS Corrector software (Makarenkov et al.,Bioinformatics 23(11):1408-0409, 2007). Data were normalized using “wellcorrection” (Makarenkov et al., Bioinformatics 23(13):1648-1657, 2007)and hit identification was achieved using clustering by “sum of theaverage squared inside-cluster distances” (Gagarin et al., J. Biomol.Screen 11(8):903-914, 2006). For hit detection, a stringent threshold(sigma 3.5) was applied to avoid false positives.

Results of HTS Growth Assay

FIG. 3 shows the results on one the plates (4143-11) included in thegrowth HTS using PAE cells. The plot represents fluorescence emission ofall 96 wells over 7 days. As expected, fluorescence values increase overtime although different wells show different fluorescence values. Acomposite image of all 96 wells is also shown (at day 4 of growth).Positive controls are shown in the far right column of wells; asexpected, these controls show maximum values of fluorescence (in theplot) and high density of cells in the wells. A negative control isshown in the left column of wells; these show low fluorescence values inthe plot and low cell density in the wells. Additionally, an example ofa compound which blocks growth of PAE is shown in the first row, fourthcolumn from the left, with low fluorescence values and few cells in thewell. Furthermore, an example of a compound which does not inhibit thegrowth of PAE is shown in the seventh row, fourth column. In this case,high fluorescence values are shown in the plot together with a highnumber of cells in the well.

Growth HTS Summary

Following the same protocol, HTS growth experiments were performed forPAE, BEC, A549 and MCF7 cells. These experiments were designed toexplore the specificity of SMs with inhibitory activity in endothelialcells as compared to tumor cells from different anatomical origins.

FIG. 4 shows a heatmap that summarizes obtained results. The heatmap wasconstructed using the function heatmap.2 of the package gplots of the Rstatistical software. Clustering was performed using Euclidean distancematrix. The X axis shows the 1974 SM tested and the Y axis representssome of the growth HTS experiments performed. For all experimentsmeasurements obtained in different days have been included and asexpected show a high degree of consistency. Dark blue cells in theheatmap represent SM with strongest growth inhibitory activity and greenand yellow SM with no activity on growth.

As expected, a small percentage of the SMs in the library had aninhibitory effect on the different cell lines. Interestingly, themajority of SMs shown to block growth of endothelial cells alsoinhibited the growth of tumor cells. Statistical analysis of these dataidentified 48 SMs which consistently inhibited the growth of endothelialcells (see TABLE 10).

Comparison of Growth Inhibitory Activity in Endothelial Cells vs. TumorCells

In the heatmap shown in FIG. 4, it is difficult to distinguish the SMswhich preferentially inhibit the growth of endothelial cells or tumorcells. In order to study this possibility, average growth activityvalues for endothelial cells and tumor cells were compared in abivariate scatterplot. Most of the SMs do not have an effect in growthin any of the cell lines tested and cluster in the center of thescatterplot (indicated within the middle-sized oval at the center of theplot). Also, most SMs with growth inhibitory activity affected withsimilar potency to tumor cells and endothelial cells and cluster in thelower left quadrant (indicated within the largest oval). Interestingly,a few SMs showed growth inhibitory activity in tumor cells but not inendothelial cells (small oval; see also TABLE 10). Growth activity ofthese small molecules is shown in the adjacent plot (small molecules areidentified by their position in the plate; TABLE 10 correlates plateposition to NSC number). No SMs were found with specific inhibitoryactivity for endothelial cells.

Specificity in the growth inhibitory activity of SMs is an importantfeature in the context of combinatorial drug therapy. In the treatmentof angiogenesis-dependent tumors it is likely important to first deliverdrugs that inhibit tumor growth but do not affect endothelial cells inorder to not damage the vasculature which serves as drug deliveryconduit to effectively reach the tumor. Therefore, drugs like the onesfound in this study with the ability of specifically inhibit tumorgrowth but not endothelial cell growth would be of great value. Once thetumor has been significantly reduced, drugs with inhibitory activity inboth tumor cells and endothelial cells would be preferred since bothtumor cells and endothelial cells need to be targeted. A number of suchdrugs have also been found in this study (see TABLE 10).

Half Maximal Inhibitory Concentration (IC₅₀) in PAE

Dose response curves were constructed for all the SM of interest usingPAE cells.

Data were fitted to non-linear sigmoid curves using GraphPad Prism(GraphPad software, Inc.). This assay was designed as a multipurposeexperiment aimed to: (1) confirm bioactivity of SMs of interest, (2)confirm dose response of the growth inhibitory activity, and (3)calculate the half maximal inhibitory concentration (IC50). Since theinitial screening was performed using a final SM concentration of 1 μM,IC50 form most compounds were confirmed to be in the range of 10⁻¹² to10⁻⁹ M (FIG. 6). This confirms that all the SMs discovered in thisproject show high growth inhibitory potency in endothelial cells (seeTABLE 10).

Cytotoxicity Assay

Evaluation of the results obtained in the HTS growth assay does notprovide information on whether the identified bioactive compoundsinhibit growth through cell toxicity. Cytotoxicity is a common problemassociated with non-peptidic small molecule drugs. In order to explorethe cytotoxic potential of the SMs of interest, a novel high throughputcytotoxicity assay was previously developed (U.S. application Ser. No.12/060,752; published as US 2009/0088341 on Apr. 2, 2009). The assay isbased on the fact that cytotoxicity involves damage to the cell membranewhich results in release of cytoplasmic content to the cell milieu.Since the reporter fluorescent cells used herein constitutivelysynthesize fluorescent proteins which are present in the cytoplasm,liberation of fluorescence to the medium can be used as an assessment ofcytotoxicity.

FIG. 7 shows two different hypothetical outcomes of the cytotoxicityassay. In the lower area of the diagram, fluorescent cells are exposedto a cytotoxic substance, which results in the liberation of florescenceto the cell culture medium. Both the fluorescence in the medium and theremaining fluorescence in the cells can be quantitated and used todetermine percentage of cytotoxicity using the formula in the lower areof the figure. The plot shows the expected dose response curve whenTriton X is used as cytotoxic agent on PAE cells.

FIG. 8 shows an example of four compounds with growth inhibitoryactivity from which two present a strong and moderate cytotoxic activityrespectively. Using this cytotoxicity assay four compounds wereidentified as cytotoxic (see TABLE 10). Although these cytotoxiccompounds may be of clinical interest, they were excluded fromconsideration for subsequent in vivo experiments.

Tube Formation

A HTS tube formation assay was also used for evaluation ofantiangiogenic compounds. After being seeded, the endothelial cells arehomogeneously distributed on the matrigel. Over time, cells migrate andinteract with other endothelial cells to form tube-like structures whichmimic the vasculature in vivo. Tube formation recapitulates several keysteps of the angiogenic process: endothelial cell activation, cellmigration, matrix degradation, cell polarization, cell to cellinteraction and tube formation (among others).

Data Analysis using AngioApplication™

The major obstacle that was encountered in adapting the tube formationassay to a HTS format was performing a morphological quantitativeanalysis of the tube formation. For that purpose, an image analysisprogram named AngioApplication™ was developed (described in detail inU.S. application Ser. No. 12/060,752; published as US 2009/0088341 onApr. 2, 2009). This software (FIG. 9) is able to rapidly assess avariety of metrics in images of tube formation including (but notlimited to) tube length, node area, branching points, fractal dimensionand lacunarity.

In order to understand which one of those metrics better explained thevariability of the HTS tube formation assay data, a principal componentanalysis (PCA) was run. The PCA showed that branching index (the numberof branches which converge in each node) and lacunarity (the averagearea of the empty spaces left by the tubes in the images) explain 53.8%and 45.0% respectively of the variability of the data (components C1 andC2 in the plots below) (see FIG. 10), making them the most appropriatemetrics to measure tube formation.

Both emptiness (C1) and branching index (C2) were plotted in a bivariatescatter plot for every SM tested. The Euclidean distance between theaverage of the positive controls and every SM was used as metric todefine anti-tube formation activity (calculations were done separatelyfor every plate). In essence, compounds which are further away from thepositive controls are more likely to be antiangiogenic. FIG. 11illustrates the results from all the SMs in one test plates. Asexpected, most of the compounds (small squares clustered in center ofgraph) are located closely to the positive controls (large squaresclustered near center of graph); this cluster is due to the fact thatmost small molecules do not have an effect on tube formation andtherefore show similar branching index and emptiness values. Incontrast, the negative controls (large squares clustered in lower rightcorner of graph) are positioned farther away from the positive controls.Representative images of the positive and negative controls are shown.Tube formation inhibitory compounds are detected as being located at anintermediate distance between the positive controls and the negativecontrols. A representative image of an active SM is shown (FIG. 11).

35 out the 1974 compounds in the library (1.75%) were found tostatistically significantly inhibit tube formation (see TABLE 10).

Dose Response of NSC 119889 in Tube Formation Assay

IC50 were calculated for all tube formation inhibitor SMs. As expected,most IC50 were in the range of 10⁻⁹ to 10⁻¹²M, making these compoundshighly effective tube formation inhibitors. FIG. 12 shows an example ofthe dose response generated with compound NSC 119889.

Results of Screen and Analysis

FIG. 13 summarizes the results obtained in the growth and tube formationHTS for endothelial cells (for information on specific compounds, seeTABLE 10). 2.4% (48) of the compounds were growth inhibitors and 1.75%(35) were tube formation inhibitors. Interestingly, 0.5% (11) of thecompounds showed both growth and tube formation inhibitory activity.These SMs are especially interesting from the perspective of networkpharmacology. It has been suggested that exquisitely selectivecompounds, compared with multitarget drugs, may exhibit lower thandesired clinical efficacy (Hopkins, Nat. Chem. Biol. 4(11):682-690,2008). However, it is challenging to design multitarget drugs whilemaintaining their drug-like properties. Here we have identified 11 SMswhich show both growth and tube formation inhibitory activity (see TABLE10).

Structure-Based Analysis

The structures of the antiangiogenic SMs identified herein were comparedwith annotated compounds in available annotated SM databases such asPubChem, DrugBank, LeadScope and FDA Marketed Drugs among others.Structural classifications were performed with LeadScope software. Onlya few of our SMs were structurally related to annotated compounds inother databases (numbers in parenthesis in FIG. 14). This can beexplained by the novel drug discovery methodology utilized in thisproject, which, as expected, results in novel SARs discoveries.Particularly interesting is the fact that none of the antiangiogenic SMsdiscovered herein are structurally related to any of the knownantiangiogenic SMs. This supports the novelty of these newly-discoveredantiangiogenic SMs and emphasizes that new SARs will result inexploitation of new cellular antiangiogenic pathways.

One of the areas for future work in this project is the identificationof specific mechanisms of action for the newly-discovered antiangiogenicSMs. Some progress has already been achieved by applying Tanimoto'ssimilarity algorithm (40-80% similarity) to compare the SMs describedherein with SMs with known mechanism of action (Fligner et al.,Technomet, 110-19, 2002. The structure of 12 compounds was found to becompatible with a potential mechanism of action (noted in parenthesis in“Mechanism of Action” in TABLE 10).

TABLE 10 BIOACTIVE SMALL MOLECULES Location NSC Growth IC50 ApoptosisMechanism Compound in Plate Numbers Bioactivity Cytotoxicity (%) (M) inPAE PAE (RFU) of Action 1 4125_G10 329226 1 7.564570142 3.719E−102.63432836 2 4127_D3 15234 1 7.734030998 1.106E−10 0.70335821 3 4127_E315226 1 9.013157895 >1.00E−06 0.81599813 4 4127_E6 24076 17.346874506 >1.00E−06 0.6823694 5 4127_E11 26081 1 7.103315772 >1.00E−060.67490672 6 4130_D6 133896 1 6.968579807 5.77E−10 0.68983209 7 4131_C11675865 1 7.322550338 1.118E−11 1.63945896 8 4131_E10 10460 18.512827087 >1.00E−06 0.85797575 9 4132_D9 207895 1 7.69900465 2.297E−100.98763993 Tubulin Binder (80) 10 4133_H4 99445 1 6.79005243 >1.00E−061.05550373 DNA synthesis inhibitor (80) 11 4133_H10 88903 1 18.697224062.366E−11 0.90298507 12 4134_A8 177407 1 6.734835623 3.016E−101.66487873 13 4135_D8 123111 1 7.062612517 1.65E−11 0.96338619Alkylating agent (40) 14 4136_F10 329261 1 6.510813456 1.885E−090.93913246 15 4137_C5 13316 1 7.93334652 6.552E−10 0.93516791 16 4137_G35844 1 6.779904279 >1.00E−06 0.90578358 17 4138_C3 5857 1 7.3449793763.979E−10 2.10704291 18 4138_E2 656202 1 7.599327541 4.635E−10 1.588619419 4138_G2 2186 1 6.564555483 9.626E−10 1.34001866 20 4138_H7 97845 18.180294654 4.684E−09 0.94986007 Tubulin binder (80) 21 4139_H6 368891 16.565516968 2.291E−11 0.96805037 22 4140_A3 126710 1 6.7452117761.535E−12 1.22807836 23 4140_C2 109836 1 7.015392379 1.207E−090.82649254 24 4140_D11 67485 1 8.537374177 >1.00E−06 1.37546642 254140_E9 47932 1 9.566749391 2.267E−10 1.43283582 Antifungal (60) 264141_F2 176327 1 8.243912666 8.379E−11 2.6798041 27 4141_F6 48630 17.89544627 >1.00E−06 0.95895522 28 4141_G8 71669 1 8.645721955 7.412E−111.34025187 29 4142_E8 150289 1 9.782711919 >1.00E−06 0.97504664 304143_A4 311153 1 8.808756865 9.265E−10 0.63899254 31 4143_A9 45238 17.617204067 0.0005062 1.23763993 32 4143_C6 156305 16.821926473 >1.00E−06 1.34981343 Topoisomerase II inhibitor (40) 334143_C9 62914 1 7.444298641 2.801E−10 2.06296642 34 4143_C10 606985 110.19900361 9.919E−12 2.93703358 Topoisomerase II inhibitor (80) 354144_G7 254681 1 7.976999744 2.41E−11 3.30060634 Topoisomerase IIinhibitor (60) 36 4145_D4 268665 1 7.657827234 2.453E−10 0.97737873 DNAsynthesis inhibitor (60) 37 4145_G5 306698 1 7.923868435 >1.00E−062.03708022 38 4121_A6 4972 2 6.742820667 0.70335821 39 4121_A8 19630 28.537524348 0.73997201 40 4123_H10 2805 2 7.23286013 0.6770056 414124_E8 16555 2 7.591147773 0.8048041 42 4125_B6 3535 2 6.9930093430.84584888 43 4127_G11 27063 2 6.439058017 1.13456157 44 4128_D5 47924 26.449536192 0.89412313 45 4129_D8 36738 2 6.610206089 0.87290112 464130_H10 108895 2 7.361554387 0.86054104 47 4131_H11 681152 26.856025461 0.59864739 48 4136_E6 632536 2 7.00534393 0.80806903 494140_G4 122351 2 6.968172168 1.23507463 50 4142_D8 268879 2 13.727913243.10284515 DNA synthesis inhibitor (40) 51 4142_H2 48458 2 8.5644439631.66954291 52 4143_E3 209910 2 9.581766236 1.22504664 53 4143_E10 3280872 8.762714287 1.21245336 54 4144_D9 521777 2 9.262232258 8.04127799 554144_D11 310551 2 28.17915266 0.92863806 56 4144_E2 292222 2 7.5205921263.41907649 Tubulin Binder (60) 57 4144_G11 321237 2 7.3772406961.14552239 DNA synthesis inhibitor (40) 58 4144_H2 259969 2 11.19182935.61497201 59 4144_H5 259968 2 9.793991594 6.21501866 60 4145_C4 2033282 6.947818406 1.26096082 61 4145_E6 166687 2 7.901324787 1.84071828 624145_H6 119889 2 13.63421931 1.26888993 63 4132_F3 676693 3 7.481198181.546E−11 1.44776119 **** 64 4135_D7 122657 3 7.092535262 2.79E−100.78941231 **** 65 4138_B4 295642 3 13.74937928 1.085E−11 2.22714552 664139_B8 13480 3 13.10907474 4.117E−10 1.04967351 67 4139_B11 150117 37.776786651 9.16E−11 0.67537313 **** 68 4139_C8 18877 3 19.126818141.404E−10 1.3542444 69 4141_B4 48300 3 8.261734079 3.837E−10 1.1354944**** 70 4142_A2 321206 3 38.52519823 6.112E−12 1.02122201 71 4142_B6292596 3 9.501696674 7.577E−11 2.30573694 **** 72 4144_G4 112200 313.88370945 1.386E−10 1.36847015 73 4145_D5 274547 3 15.663006443.579E−10 0.90625 74 4123_B6 4265 4 75 4130_G5 130830 4 76 4133_D1154044 4 77 4143_A5 327705 4 Bioactivity 1 Endothelial Cell GrowthInhibitor 2 Tube Formation Inhibitor 3 Growth Inhibitor + Tube FormationInhibitor 4 Specific Tumor Cell Growth Inhibitor **** Compounds withboth growth and tube formation inhibitory activities and no significantcytotoxicity.

Example 2 In Vivo Inhibition of Angiogenesis in Xenograft Tumors

This example shows the in vivo inhibition of angiogenesis in tumorxenografts by administration of selected small molecules describedherein.

Methods

For generation of mouse xenografts, female athymic nude mice wereinjected with 5×10⁶ A549 or SK-ML-1 cells (100 μl/mouse) in the lefthindquarters. The resulting tumors were measured three times a week andbody weight was measured twice a week. 14 days following tumor cellinjection, mice with tumor burdens greater than 100 mm³ or less than 50mm³ were eliminated from the study. The remaining mice were randomizedinto groups (10 animals per group) and treated three times a week(Mon/Wed/Fri) for four weeks with 100 of 10 μM sterile drug solutions(stored at 4° C.) that were administered via IP injection. Tumors weremeasured three times per week (Mon/Wed/Fri) for four additional weeksand mice weighed twice weekly (Tues/Thu) for an additional four weeks.On week six or when tumors exceeded 2 cm, the mice were euthanized. Afull necropsy was performed and any abnormal tissues were snap frozen(−80° C.). Tumors were excised and bisected into four parts. Two partswere fixed in 2% formalin overnight at 4° C., rinsed in cold PBS andprepared for paraffin embedding. The other two parts were snap frozen ondry ice or liquid nitrogen and stored at −80° C.

Results

7 of the 77 SMs described above were chosen for in vivo xenograftexperiments. SMs were selected based on the type of inhibition (tubeformation-NSC 19630, NSC 292222, NSC 259969; or tubeformation+growth-NSC 122657, NSC 150117, NSC 48300, NSC 292596),percentage of inhibition, low cytotoxicity levels and availability. Forthe tumor xenografts, two different human cancer models were chosen.A549 is a lung carcinoma which induces almost exclusively peritumoralvasculature in subcutaneous tumors. In contrast, SK-ML-1 is aleiomyosarcoma which induces high levels of intratumoral angiogenesis.Every experiment included a negative PBS control as well as the knownantiangiogenic drug AVASTIN® as a positive control. AVASTIN® waspreviously shown to significantly inhibit the growth of both A549 aswell as SK-ML-1.

As shown in FIG. 15, all the small molecules inhibited tumor growth tovarying degrees. In general, and as expected, the small molecules morestrongly inhibited the growth of the angiogenic tumor SK-ML-1 (bottompanels) in comparison to the less angiogenic tumor A549 (top panels). Ofthe SMs tested, the strongest inhibitors of tumor growth were NSC 48300,NSC 150117 and NSC 259969, all of which showed potencies similar toAVASTIN® in the SK-ML-1 model.

Example 3 Tubulin Binding Potential of Antiangiogenic Small Molecules

Many known antiangiogenic drugs bind to tubulin and interfere with itspolymerization. Likewise, it has also been shown that molecules whichinterfere with tubulin polymerization are potentially antiangiogenic. Incontrast, the above-described SAR analysis predicted that none of theseven small molecules shown in Example 2 to be antiangiogenic inxenograft assays would inhibit tubulin polymerization. This exampleconfirms this prediction.

The tubulin binding activity of the small molecules studied in vivo wascharacterized using a fluorescence-based, tubulin polymerization assayfrom Cytoskeleton (Denver, Colo.; Cat. # BK011P), according to themanufacturer's instructions. As predicted by the SAR analysis, none ofthe small molecules studied interfered with tubulin polymerization (FIG.16).

Example 4 Effect of SMs on Receptor Tyrosine Kinase Activity

Most of the currently FDA approved antiangiogenic therapies (such asAVASTIN® or sunitinib) target receptor tyrosine kinase (RTK) activity.It has recently been proposed by independent groups (Pàez-Ribes et al.,Cancer Cell, 15: 220-231, 2009; Ebos et al., Cancer Cell, 15: 232-239,2009) that RTK inhibitors have deleterious collateral effects, includingstimulation of metastasis and alternative angiogenesis pathways otherthan those inhibited by the drugs. This example shows thecharacterization of the RTK inhibitory activity of a subset of the smallmolecules described herein.

To characterize the RTK inhibitory activity of the antiangiogenic SMsdescribed herein, 36 compounds were chosen and screened usingInvitrogen's SelectScreen® kinase activity profiling service (describedon-line at tools.invitrogen.com/content.cfm?pageid=10413#selection).VEGFR1 and FGFR2 RTK inhibitory activities were studied. The results ofthe screen are detailed in Table 11. The % inhibition (and mean %inhibition) of RTK activity from two independent trials is shown Of theseven SMs tested in vivo in Example 2, only NSC 19630 and NSC 48300showed RTK inhibitory activity, and only for for the VEGFR2 receptor.Overall, a small minority of the SMs tested had any substantial kinaseinhibitory activity. This observation supports a mechanism ofantiangiogenic action other than RTK inhibition for most of the SMsdescribed herein.

TABLE 11 KINASE INHIBITION Comp. Kinase Activity % % Mean % NSC# TestedInhibition Inhibition Inhibition 4972 FGFR1 3 −7 −2 4972 KDR(VEGFR2) 211 7 19630 FGFR1 2 −2 0

2805 FGFR1 1 10 6 2805 KDR(VEGFR2) 42 52 47 16555 FGFR1 3 3 3 16555KDR(VEGFR2) 11 11 11 3535 FGFR1 −1 −3 −2 3535 KDR(VEGFR2) 10 5 8 27063FGFR1 −3 −2 −3 27063 KDR(VEGFR2) 12 11 11 47924 FGFR1 1 3 2 47924KDR(VEGFR2) 21 18 19 36738 FGFR1 −1 4 1 36738 KDR(VEGFR2) 16 17 16108895 FGFR1 4 4 4 108895 KDR(VEGFR2) 5 6 6 681152 FGFR1 4 3 3 681152KDR(VEGFR2) 10 8 9 676693 FGFR1 4 3 3 676693 KDR(VEGFR2) 1 8 5 122657FGFR1 −3 −3 −3 122657 KDR(VEGFR2) 14 14 14 632536 FGFR1 1 1 4 632536KDR(VEGFR2) 15 16 16 295642 FGFR1 2 9 6 295642 KDR(VEGFR2) 19 17 18150117 FGFR1 2 7 5 150117 KDR(VEGFR2) 28 24 26 13480 FGFR1 5 10 8 13480KDR(VEGFR2) 4 2 3 18877 FGFR1 −3 0 −2 18877 KDR(VEGFR2) 10 4 7 122351FGFR1 −1 4 1 122351 KDR(VEGFR2) 4 4 4 48300 FGFR1 8 10 9

321206 FGFR1 5 5 5 321206 KDR(VEGFR2) 52 45 49 292596 FGFR1 4 4 4 292596KDR(VEGFR2) 9 18 14 268879 FGFR1 3 −2 1 268879 KDR(VEGFR2) 40 38 3948458 FGFR1 5 8 6 48458 KDR(VEGFR2) 13 9 11 328087 FGFR1 3 5 4 328087KDR(VEGFR2) 9 9 9 209910 FGFR1 2 5 4 209910 KDR(VEGFR2) 9 10 10 310551FGFR1 −5 −1 −3 310551 KDR(VEGFR2) 21 18 20 521777 FGFR1 1 0 1 521777KDR(VEGFR2) 10 8 9 292222 FGFR1 2 5 3 292222 KDR(VEGFR2) 8 7 7 321237FGFR1 1 5 6 321237 KDR(VEGFR2) 105 104 105 112200 FGFR1 9 5 7 112200KDR(VEGFR2) 100 99 99 259969 FGFR1 3 3 3 259969 KDR(VEGFR2) 0 9 5 259968FGFR1 5 2 3 259968 KDR(VEGFR2) 8 9 8 203328 FGFR1 4 4 4 203328KDR(VEGFR2) 9 7 8 274547 FGFR1 4 7 6 274547 KDR(VEGFR2) 14 12 13 166687FGFR1 21 22 22 166687 KDR(VEGFR2) 9 6 8 119889 FGFR1 96 95 96 119889KDR(VEGFR2) 103 104 103 SMs that inhibited kinase activity 40% or moreare indicated in bold. SMs also characterized for in vivo activity areitalicized.

Example 5 Effect of Antiangiogenic SMs on Gene Expression DuringEndothelial Tube Formation

This example shows the effect of anti-angiogenic small moleculesdescribed herein on the expression of genes in the angiogenesis pathway.

Methods

For gene expression studies, three independent experiments were run foreach SM tested. 90.000 cells/well (6 wells/treatment) of dermalmicrovascular endothelial cells (Lonza, Walkersville, Md.) were seededon polymerized GELTREX™ gel matrix (Invitrogen, Carlsbad, Calif.) in 24well plates. Wells were immediately treated with the same volume of 1 μMSM or PBS. After 24 hours incubation at 37° C. and 5% CO₂, cells wereextracted using Cell Recovery Solution (BD Biosciences, San Jose,Calif., Cat. #354253). Total RNA was extracted with the RNeasy Mini Kit(Qiagen, Valencia, Calif., Cat. #74104), and retrotranscribed usingSUPERSCRIPT® First-Strand reverse transcriptase (Invitrogen, Carlsbad,Calif., Cat. #11904-018). The real time PCR reactions were performed inan Opticon 2 cycler (MJ Research, Waltham, Mass.). Amplification wasperformed in a final volume of 25 μl, containing 2 μl cDNA (1:10dilution from the reversed transcribed reaction) and 2 μl of primermixture (10 μM each of forward and reverse primers). Samples wereamplified as follows: after initial denaturation at 95 C for 2 minutes,reactions were run for 46 cycles at 95° C. for 30 seconds, 60° C. for 30seconds, and 72° C. for 45 seconds. Fluorescence was measured in everycycle and a melting curve was run after the PCR by increasingtemperature from 50 to 96° C. (in 0.5° C. increments). A defined singlepeak was obtained for all amplicons, thus confirming the specificity ofthe amplification.

Results

To better understand the mechanism of action of the seven smallmolecules used in the in vivo studies presented in Example 2, the effectof these molecules on the expression of angiogenesis genes in primaryendothelial cells during tube formation was studied. The genes that weremonitored were selected based on their relevance to the angiogenesisprocess. The genes assayed and primers used in the real-time PCR arepresented in Tables 12 and 13. All experiments were done in triplicate.The results are shown in the volcano plots of FIG. 17. Log 2 fold-changeis presented in the X-axis and log 10 P value in the Y-axis. Theseresults are also summarized below in Table 14.

TABLE 12 GENES IN REAL TIME PCR ARRAYS GenBank NCBI Protein Coding DNAGene Symbol Accession Accession Length AKT1 NM_005163 NP_005154 1443ANGPT1 NM_001146 NP_001137 1497 ANGPT2 AF187858 AAF76526 1335 ANGPTL3NM_014495 NP_055310 1383 ANGPTL4 NM_016109 NP_057193 1221 ANPEPNM_001150 NP_001141 2904 BAI1 NM_001702 NP_001693 4755 CCL11 NM_002986NP_002977 294 CCL2 NM_002982 NP_002973 300 CDH5 NM_001795 NP_001786 2355COL18A1 NM_030582 NP_085059 4551 COL4A3 NM_000091 NP_000082 5013 dll4NM_019074 NP_061947 2058 CXCL10 NM_001565 NP_001556 297 CXCL3 NM_002090NP_002081 321 CXCL5 NM_002994 NP_002985 345 CXCL6 NM_002993 NP_002984345 CXCL9 NM_002416 NP_002407 378 TYMP NM_001953 NP_001944 1449 S1PR1NM_001400 NP_001391 1149 EFNA1 NM_182685 NP_872626 552 EFNA3 NM_004952NP_004943 717 EFNB2 NM_004093 NP_004084 1002 EGF NM_001963 NP_0019543624 ENG NM_000118 NP_000109 1878 EPHB4 NM_004444 NP_004435 2964 EREGNM_001432 NP_001423 510 FGF1 NM_000800 NP_000791 468 FGF2 NM_002006NP_001997 633 FGFR3 NM_000142 NP_000133 2421 FIGF NM_004469 NP_0044601065 FLT1 NM_002019 NP_002010 4017 HAND2 NM_021973 NP_068808 654 HGFNM_000601 NP_000592 2187 HIF1A NM_001530 NP_001521 2481 HPSE NM_006665NP_006656 1632 ID1 NM_002165 NP_002156 468 ID3 NM_002167 NP_002158 360IFNA1 NM_024013 NP_076918 570 IFNB1 NM_002176 NP_002167 564 IFNGNM_000619 NP_000610 501 IGF1 NM_000618 NP_000609 462 IL1B NM_000576NP_000567 810 IL6 NM_000600 NP_000591 639 IL8 NM_000584 NP_000575 300ITGAV NM_002210 NP_002201 3147 ITGB3 NM_000212 NP_000203 2367 JAG1NM_000214 NP_000205 3657 KDR NM_002253 NP_002244 4071 LAMA5 NM_005560NP_005551 11088 LECT1 NM_007015 NP_008946 1005 LEP NM_000230 NP_000221504 MDK NM_002391 NP_002382 432 MMP2 NM_004530 NP_004521 1983 MMP9NM_004994 NP_004985 2124 Notch4 NM_004557 NP_004548 6009 NRP1 NM_003873NP_003864 2772 NRP2 NM_003872 NP_003863 2781 PDGFA NM_002607 NP_002598636 PECAM1 NM_000442 NP_000433 2217 PGF NM_002632 NP_002623 513 PLAUNM_002658 NP_002649 1296 PLG NM_000301 NP_000292 2433 PLXDC1 NM_020405NP_065138 1503 PROK2 NM_021935 NP_068754 327 PTGS1 NM_000962 NP_0009531800 SERPINF1 NM_002615 NP_002606 1257 SPHK1 NM_021972 NP_068807 1197STAB1 NM_015136 NP_055951 7713 TEK NM_000459 NP_000450 3375 TGFANM_003236 NP_003227 483 TGFB1 NM_000660 NP_000651 1176 TGFB2 NM_003238NP_003229 1245 TGFBR1 NM_004612 NP_004603 1512 THBS1 NM_003246 NP_0032373513 THBS2 NM_003247 NP_003238 3519 TIMP1 NM_003254 NP_003245 624 TIMP2NM_003255 NP_003246 663 TIMP3 NM_000362 NP_000353 636 TNF NM_000594NP_000585 702 TNFAIP2 NM_006291 NP_006282 1965 VEGFA NM_003376 NP_003367648 VEGFC NM_005429 NP_005420 1260 CD248 NM_020404 NP_065137 2274 GPR124AB040964 BAA96055 3621 PLXDC1 NM_020405 NP_065138 1503 ANTXR1 NM_032208NP_115584 1695 RASD2 NM_014310 NP_055125 801 ARHGEF17 NM_014786NP_055601 6192 TNS3 AL833845 CAD38705 2714 DKK3 NM_015881 NP_056965 1053MMP11 NM_005940 NP_005931 1467 NID1 BC045606 AAH45606 3345 THY1NM_006288 NP_006279 486 CST4 NM_001899 NP_001890 426 MRC2 NM_006039NP_006030 4440 TNS1 AK001785 BAA91910 1197 BMP1 AF318323 AAL55830 933COMT NM_000754 NP_000745 816 PTPRCAP NM_005608 NP_005599 621 57722AB046848 BAB 13454 2943 EXTL3 NM_001440 NP_001431 2760 vWF NM_000552NP_000543 8442 PNMT NM_002686 NP_002677 849 58488 BC005112 AAH05112 582ltbp4 AK074499 BAC11024 1911 23001 AK055806 BAB71020 2169  4247NM_002408 NP_002399 1344 Sdc4 NM_002999 NP_002990 597 Ralb NM_002881NP_002872 621 DGKG NM_001346 NP_001337 2376 TRA2B AK098191 BAC05256 759CLIC1 NM_001288 NP_001279 726 90780 NM_138300 NP_612157 1221  5569NM_181839 NP_862822 231 151516  NM_152792 NP_690005 1032 CALD1 AF247820AAF69498 1446 DUT AF018432 AAB71393 759 copa BC038447 AAH38447 3702PDCD2 NM_144781 NP_659005 687 151516  NM_152792 NP_690005 1032 VGLL4D50911 BAA09470 891 APOE NM_000041 NP_000032 954  8131 NM_012075NP_036207 1710 10988 AK091730 BAC03733 1368  9569 NM_005685 NP_0056762835 Acvrl1 NM_000020 NP_000011 1512 Adcy4 NM_139247 NP_640340 3234Calcrl NM_005795 NP_005786 1386 Caskin2 NM_020753 NP_065804 3609 Ccbp2NM_001296 NP_001287 1155 Cldn5 BC019290 AAH19290 801 01839 NM_001945NP_001936 627 Egfl7 NM_016215 NP_057299 822 Ehd4 NM_139265 NP_6446701626 Entpd1 AJ133134 CAB41887 921 Epas1 BC015869 AAH15869 306 ErgNM_004449 NP_004440 1389 ESAM1 NM_138961 NP_620411 1173 Fgd5 BX640820CAE45896 3387 Gpr116 AL050295 CAB43394 1855 Hspa12b NM_052970 NP_4432022061 Icam1 NM_000201 NP_000192 1599 Icam2 NM_000873 NP_000864 828 Kifc1BC000712 AAH00712 2180 Lats2 NM_014572 NP_055387 3267 Lrrk1 AB058693BAB47419 4112 Mmrn2 NM_024756 NP_079032 2850 Myo1b AJ001381 CAA047121310 PALD AB033100 BAA86588 2586 NM_023516 BC015770 AAH15770 1446 55332BC018435 AAH18435 717 CTTNBP2NL NM_018704 NP_061174 1920 CENTD3NM_022481 NP_071926 4635 C1orf54 NM_024579 NP_078855 396 134265 AK074185 BAB85011 2381 Npr3 NM_000908 NP_000899 1623 Pltp NM_182676NP_872617 1326 Ptprb BC051329 AAH51329 2316 Ptprm NM_002845 NP_0028364359 GRRP1 BC025658 AAH25658 816 stard9 AB037721 BAA92538 5464 Ramp2NM_005854 NP_005845 528 Rasip1 BC042111 AAH42111 1567 Robo4 AK074163BAB84989 2109 Sdpr NM_004657 NP_004648 1278 Slc43a3 AP118070 AAF22014318 Slc9a3r2 U82108 AAB53042 981 Slco2a1 BC041140 AAH41140 2035 B2MNM_004048 NP_004039 360 HPRT1 NM_000194 NP_000185 657 RPL13A NM_012423NP_036555 612 GAPDH NM_002046 NP_002037 1008 ACTB NM_001101 NP_0010921128

TABLE 13 REAL TIME PCR PRIMERS PrimerBank Gene ID Forward PrimerReverse Primer AKT1  4885061a1 GCACAAACGAGGGGAGTACAT CCTCACGTTGGTCCACATC(SEQ ID NO: 1) (SEQ ID NO: 2) ANGPT1 20532340a1 CTCGCTGCCATTCTGACTCACGACAGTTGCCATCGTGTTCTG (SEQ ID NO: 3) (SEQ ID NO: 4) ANGPT2  8570647a1TCTTGGCCGCAGCCTATAAC TGCTGGACCTGATATTGCTTCT (SEQ ID NO: 5)(SEQ ID NO: 6) ANGPTL3  7656888a1 CTTCAATGAAACGTGGGAGAACTGCCAGTAATCGCAACTAGATGT (SEQ ID NO: 7) (SEQ ID NO: 8) ANGPTL4 21536396a1TCCTGGGACGAGATGAATGTC CTGAGCCTTGAGTTGTGTCTG (SEQ ID NO: 9)(SEQ ID NO: 10) ANPEP  4502095a1 GCACAATCATCGCACTGTCAGCGCTTTACTTTGGTCCAAGGT (SEQ ID NO: 11) (SEQ ID NO: 12) BAI1  4502355a1GCGGCGCTACACTCTCTAC AGCACCTCGTCGAAGCTCT (SEQ ID NO: 13) (SEQ ID NO: 14)CCL11  4506827a1 ATACCCCTTCAGCGACTAGAG GCTTTGGAGTTGGAGATTTTTGG(SEQ ID NO: 15) (SEQ ID NO: 16) CCL2  4506841a1 CAGCCAGATGCAATCAATGCCTGGAATCCTGAACCCACTTCT (SEQ ID NO: 17) (SEQ ID NO: 18) CDH5  4502727a1GATCAAGTCAAGCGTGAGTCG AGCCTCTCAATGGCGAACAC (SEQ ID NO: 19)(SEQ ID NO: 20) COL18A1 13385620a1 GCTGAACCTGAACTGGCTTTGGACACCGGCAATGTTCTCCTC (SEQ ID NO: 21) (SEQ ID NO: 22) COL4A3 10835113a1CAGCTCTGATGCCAATGAACA TTGCACGTTCCTCTTCCATGA (SEQ ID NO: 23)(SEQ ID NO: 24) dll4  9506545a1 TCCAACTGCCCTTCAATTTCACCTGGATGGCGATCTTGCTGA (SEQ ID NO: 25) (SEQ ID NO: 26) CXCL10  4504701a1GTGGCATTCAAGGAGTACCTC GCCTTCGATTCTGGATTCAGACA (SEQ ID NO: 27)(SEQ ID NO: 28) CXCL3  4504157a1 CGCCCAAACCGAAGTCATAGGCTCCCCTTGTTCAGTATCTTTT (SEQ ID NO: 29) (SEQ ID NO: 30) CXCL5  4506849a1GAGAGCTGCGTTGCGTTTG TTTCCTTGTTTCCACCGTCCA (SEQ ID NO: 31)(SEQ ID NO: 32) CXCL6  4506851a1 AGAGCTGCGTTGCACTTGTTGCAGTTTACCAATCGTTTTGGGG (SEQ ID NO: 33) (SEQ ID NO: 34) CXCL9  4505187a1CCAGTAGTGAGAAAGGGTCGC TGGGGCAAATTGTTTAAGGTCTT (SEQ ID NO: 35)(SEQ ID NO: 36) TYMP  4503445a1 AGCTGGAGTCTATTCCTGGATTGGCTGCATATAGGATTCCGTC (SEQ ID NO: 37) (SEQ ID NO: 38) S1PR1 13027636a1CTTGCTGACCATTTGGAAAACC CTGTGTAGGCTACTCCTGCC (SEQ ID NO: 39)(SEQ ID NO: 40) EFNA1 33359680a1 CGGAGAAGCTGTCTGAGAAGTCTGAGGACTGTGAGAGATGTAGT (SEQ ID NO: 41) (SEQ ID NO: 42) EFNA3  4826708a1TCTCTGGGCTACGAGTTCCAC ACGTTGATCTTCACATTGGGG (SEQ ID NO: 43)(SEQ ID NO: 44) EFNB2  4758250a1 ACTGCTGGGGTGTTTTGATGGTGTGGGTATAGTACCAGTCCTTG (SEQ ID NO: 45) (SEQ ID NO: 46) EGF  4503491a1AAGGTACTCTCGCAGGAAATGG ACATACTCTCTCTTGCCTTGACC (SEQ ID NO: 47)(SEQ ID NO: 48) ENG  4557555a1 AGCCCCACAAGTCTTGCAGGCTAGTGGTATATGTCACCTCGC (SEQ ID NO: 49) (SEQ ID NO: 50) EPHB4 32528301a1CGGCAGCCTCACTACTCAG TCCCATTTTGATGGCCCGAAG (SEQ ID NO: 51)(SEQ ID NO: 52) EREG  4557567a1 CTGCCTGGGTTTCCATCTTCTGCCATTCATGTCAGAGCTACACT (SEQ ID NO: 53) (SEQ ID NO: 54) FGF1  4503697a1ACACCGACGGGCTTTTATACG CCCATTCTTCTTGAGGCCAAC (SEQ ID NO: 55)(SEQ ID NO: 56) FGF2 15451898a1 AGAAGAGCGACCCTCACATCAACTGCCCAGTTCGTTTCAGTG (SEQ ID NO: 57) (SEQ ID NO: 58) FGFR3  4503711a1TCCTTGCACAACGTCACCTTT GCAGAGTGATGAGAAAACCCAA (SEQ ID NO: 59)(SEQ ID NO: 60) FIGF  4758378a1 ACAGAGAGTGGGTAGTGGTGAGTTCCTCCAAACTAGAAGCAGC (SEQ ID NO: 61) (SEQ ID NO: 62) FLT1  4503749a1CTGTCATGCTAATGGTGTCCC TGCTGCTTCCTGGTCCTAAAATA (SEQ ID NO: 63)(SEQ ID NO: 64) HAND2 12545384a1 ATGAGTCTGGTAGGTGGTTTTCCCATACTCGGGGCTGTAGGACA (SEQ ID NO: 65) (SEQ ID NO: 66) HGF 33859835a1TACAGGGGCACTGTCAATACC GGATACTGAGAATCCCAACGC (SEQ ID NO: 67)(SEQ ID NO: 68) HIF1A  4504385a1 GGCGCGAACGACAAGAAAAAGCCTTATCAAGATGCGAACTCACA (SEQ ID NO: 69) (SEQ ID NO: 70) HPSE  5729873a1TCCTGCGTACCTGAGGTTTG CAACCGTAACTTCTCCTCCAC (SEQ ID NO: 71)(SEQ ID NO: 72) ID1 31317299a1 ACGAGCAGCAGGTAAACGTGGAAGGTCCCTGATGTAGTCGAT (SEQ ID NO: 73) (SEQ ID NO: 74) ID3 32171182a1AGTCCCGAGAGGCACTCAG GCTCCTTTTGTCGTTGGAGATG (SEQ ID NO: 75)(SEQ ID NO: 76) IFNA1 13128950a1 GCCTCGCCCTTTGCTTTACTCTGTGGGTCTCAGGGAGATCA (SEQ ID NO: 77) (SEQ ID NO: 78) IFNB1  4504603a1ATGACCAACAAGTGTCTCCTCC GCTCATGGAAAGAGCTGTAGTG (SEQ ID NO: 79)(SEQ ID NO: 80) IFNG 10835171a1 CTCTTGGCTGTTACTGCCAGGCTCCACACTCTTTTGGATGCT (SEQ ID NO: 81) (SEQ ID NO: 82) IGF1 11024682a1ATGCTCTTCAGTTCGTGTGTG GCACTCCCTCTACTTGCGTTC (SEQ ID NO: 83)(SEQ ID NO: 84) IL1B 10835145a1 CTCGCCAGTGAAATGATGGCTGTCGGAGATTCGTAGCTGGAT (SEQ ID NO: 85) (SEQ ID NO: 86) IL6 10834984a1AAATTCGGTACATCCTCGACGG GGAAGGTTCAGGTTGTTTTCTGC (SEQ ID NO: 87)(SEQ ID NO: 88) IL8 10834978a1 TTTTGCCAAGGAGTGCTAAAGAAACCCTCTGCACCCAGTTTTC (SEQ ID NO: 89) (SEQ ID NO: 90) ITGAV  4504763a1TCGGGACTCCTGCTACCTC CACGAGAAGAAACATCCGGGA (SEQ ID NO: 91)(SEQ ID NO: 92) ITGB3  4557677a1 AGGATGACTGTGTCGTCAGATGGTAGACGTGGCCTCTTTATACA (SEQ ID NO: 93) (SEQ ID NO: 94) JAG1  4557679a1TCGGGTCAGTTCGAGTTGGA AGGCACACTTTGAAGTATGTGTC (SEQ ID NO: 95)(SEQ ID NO: 96) KDR 11321597a1 GGCCCAATAATCAGAGTGGCATGTCATTTCCGATCACTTTTGGA (SEQ ID NO: 97) (SEQ ID NO: 98) LAMA5 21264602a1CCCACCGAGGACCTTTACTG GGTGTGCCTTGTTGCTGTT (SEQ ID NO: 99)(SEQ ID NO: 100) LECT1  5901932a1 GGTGGGACCTGATGACGTGAGCTCCCGAAATGAGGACCA (SEQ ID NO: 101) (SEQ ID NO: 102) LEP  4557715a1GAACCCTGTGCGGATTCTTGT TCCATCTTGGATAAGGTCAGGAT (SEQ ID NO: 103)(SEQ ID NO: 104) MDK  4505135a1 CGCGGTCGCCAAAAAGAAAG CAGTCGGCTCCAAACTCCT(SEQ ID NO: 105) (SEQ ID NO: 106) MMP2 11342666a1 CCGTCGCCCATCATCAAGTTCTGTCTGGGGCAGTCCAAAG (SEQ ID NO: 107) (SEQ ID NO: 108) MMP9  4826836a1TGGCAGAGATGCGTGGAGA GGCAAGTCTTCCGAGTAGTTTT (SEQ ID NO: 109)(SEQ ID NO: 110) Notch4 27894370a1 GGGTGAGACGTGCCAGTTTCCTGGGTGTCAATGGAGAGGGA (SEQ ID NO: 111) (SEQ ID NO: 112) NRP1  4505457a1TGGGGCTCTCACAAGACCTT AGCTTGGGAATAGATGAAGTTGC (SEQ ID NO: 113)(SEQ ID NO: 114) NRP2  4505459a1 GAAGGGAACATGCACTATGACAAGCGTTTTTACCGTGGGCTT (SEQ ID NO: 115) (SEQ ID NO: 116) PDGFA 15208658a1CCAGCGACTCCTGGAGATAGA CTTCTCGGGCACATGCTTAGT (SEQ ID NO: 117)(SEQ ID NO: 118) PECAM1 21314617a1 AACAGTGTTGACATGAAGAGCCTGTAAAACAGCACGTCATCCTT (SEQ ID NO: 119) (SEQ ID NO: 120) PGF 20149543a1TGCTGCGGCGATGAGAATC GTCTCCTCCTTTCCGGCTT (SEQ ID NO: 121)(SEQ ID NO: 122) PLAU  4505863a1 GTGAGCGACTCCAAAGGCAGCAGTTGCACCAGTGAATGTT (SEQ ID NO: 123) (SEQ ID NO: 124) PLG  4505881a1CAGGGGGCTTCACTGTTCAG GCCATTATCACACATTGTTGCTC (SEQ ID NO: 125)(SEQ ID NO: 126) PLXDC1 15011862a1 CCTGGGCATGTGTCAGAGCGGTGTTGGAGAGTATTGTGTGG (SEQ ID NO: 127) (SEQ ID NO: 128) PROK217530787a1 GTGACAAGGACTCCCAATGTG TCTTGACCCAGATACTGACAGC (SEQ ID NO: 129)(SEQ ID NO: 130) PTGS1 18104967a1 CTCCCAGGAGTACAGCTACGACCAGCAATCTGGCGAGAGA (SEQ ID NO: 131) (SEQ ID NO: 132) SERPINF134098938a1 GCCCTGGTGCTACTCCTCT CAGCTTGTTCACGGGGACTTT (SEQ ID NO: 133)(SEQ ID NO: 134) SPHK1 21361088a1 AGGCTGAAATCTCCTTCACGCGTCTCCAGACATGACCACCAG (SEQ ID NO: 135) (SEQ ID NO: 136) STAB1 12225240a1ACATCTGCTCGAACCCAAACA GACAGCGACATCTGGCAACA (SEQ ID NO: 137)(SEQ ID NO: 138) TEK  4557869a1 TGCCACCCTGGTTTTTACGGTTGGAAGCGATCACACATCTC (SEQ ID NO: 139) (SEQ ID NO: 140) TGFA  4507461a1GGCCCTGGCTGTCCTTATC AGCAAGCGGTTCTTCCCTTC (SEQ ID NO: 141)(SEQ ID NO: 142) TGFB1 10863873a1 GGCCAGATCCTGTCCAAGCGTGGGTTTCCACCATTAGCAC (SEQ ID NO: 143) (SEQ ID NO: 144) TGFB2  4507463a1CTGCATCTGGTCACGGTCG CCTCGGGCTCAGGATAGTCT (SEQ ID NO: 145)(SEQ ID NO: 146) TGFBR1  4759226a1 ACGGCGTTACAGTGTTTCTGGCACATACAAACGGCCTATCT (SEQ ID NO: 147) (SEQ ID NO: 148) THBS1  4507485a1TGCCTGATGACAAGTTCCAAG CCAGAGTGGTCTTTCCGCTC (SEQ ID NO: 149)(SEQ ID NO: 150) THBS2  4507487a1 ACAAAGACACGACCTTCGACCGACTTGCCGTCCTGCTTGA (SEQ ID NO: 151) (SEQ ID NO: 152) TIMP1  4507509a1CTTCTGCAATTCCGACCTCGT CCCTAAGGCTTGGAACCCTTT (SEQ ID NO: 153)(SEQ ID NO: 154) TIMP2  4507511a1 AAGCGGTCAGTGAGAAGGAAGTCCTCTTGATAGGGTTGCCATA (SEQ ID NO: 155) (SEQ ID NO: 156) TIMP3 4507513a1 CAACTCCGACATCGTGATCCG GAAGCCTCGGTACATCTTCATC (SEQ ID NO: 157)(SEQ ID NO: 158) TNF 25952111a1 ATGAGCACTGAAAGCATGATCCGAGGGCTGATTAGAGAGAGGTC (SEQ ID NO: 159) (SEQ ID NO: 160) TNFAIP226051240a1 TCCCCGAGAGCGTCTTTCT ATGTCATTGGGGTAGAGGTTCT (SEQ ID NO: 161)(SEQ ID NO: 162) VEGFA 30172564a1 CAACATCACCATGCAGATTATGCGCTTTCGTTTTTGCCCCTTTC (SEQ ID NO: 163) (SEQ ID NO: 164) VEGFC  4885653a1CACGGCTTATGCAAGCAAAGA TCCTTTCCTTAGCTGACACTTGT (SEQ ID NO: 165)(SEQ ID NO: 166) CD248  9966885a1 TGCGAACACGAATGTGTGGACAATCTGGCACTCATCTGTGTC (SEQ ID NO: 167) (SEQ ID NO: 168) GPR12420521932a1 TGAGCAATAACAAGATCACGGG TCGGAGGTGAGACAGCCAA (SEQ ID NO: 169)(SEQ ID NO: 170) PLXDC1 15011862a1 CCTGGGCATGTGTCAGAGCGGTGTTGGAGAGTATTGTGTGG (SEQ ID NO: 171) (SEQ ID NO: 172) ANTXR114149904a1 CGGTAGACGCCTCTTATTATGGT CCTTTTCCAACTTAGCACCTTCT(SEQ ID NO: 173) (SEQ ID NO: 174) RASD2 22027486a1 CAGTGTGCCCGCCAAAAACTGGGTGTGTACTGGTCCTCAA (SEQ ID NO: 175) (SEQ ID NO: 176) ARHGEF1721361458a1 CGACTCTGAATCCCCAGGAAC CCTGCGGTTGGGAGAAGATA (SEQ ID NO: 177)(SEQ ID NO: 178) TNS3 21739317a1 GGCATTACCCCGTGAACAGTCACCCCGATGTCTCTGTGAT (SEQ ID NO: 179) (SEQ ID NO: 180) DKK3 27735014a1TGGGGTCACTGCACCAAAAT GAAGGTCGGCTTGCACACATA (SEQ ID NO: 181)(SEQ ID NO: 182) MMP11  5174581a1 GAGGCCCTAAAGGTATGGAGCCCCTTCTCGGTGAGTCTTGG (SEQ ID NO: 183) (SEQ ID NO: 184) NID1 28374139a1CACATTGAGCCCTACACGGAG GCTGAGAGCATAGCGCAAGAT (SEQ ID NO: 185)(SEQ ID NO: 186) THY1 19923362a1 TCGCTCTCCTGCTAACAGTCTCTCGTACTGGATGGGTGAACT (SEQ ID NO: 187) (SEQ ID NO: 188) CST4  4503109a1CCTCTGTGTACCCTGCTACTC CTTCGGTGGCCTTGTTGTACT (SEQ ID NO: 189)(SEQ ID NO: 190) MRC2  5174485a1 CCGAAACCGGCTATTCAACCTCAGCGAAGATTCAGTGCTTCC (SEQ ID NO: 191) (SEQ ID NO: 192) TNS1 13624033a1TAGATGGGAGCCTGTATGCTAAG GTAGGACGTGTGGCATTAACA (SEQ ID NO: 193)(SEQ ID NO: 194) BMP1 18027738a1 CTCTCTCGTTTCAGAAAAGAGGCTTCCTGAGTAACAAGGGGTCC (SEQ ID NO: 195) (SEQ ID NO: 196) COMT  4502969a1TACTGCGAGCAGAAGGAGTG CCAGCGAAATCCACCATCC (SEQ ID NO: 197)(SEQ ID NO: 198) PTPRCAP  5032005a1 AGCTGGGGTCCACAGACAAGACGCCTCTCCACATTGCT (SEQ ID NO: 199) (SEQ ID NO: 200)  57722 10047333a1GCGAGCAGATCATCGGCTT TGCAAACTGGTATTCCACATTGT (SEQ ID NO: 201)(SEQ ID NO: 202) EXTL3  4503617a1 CGCTCATCGCCCACTATTACCTGTTCAGCTCTTGGCGCTT (SEQ ID NO: 203) (SEQ ID NO: 204) vWF  4507907a1AGCCTTGTGAAACTGAAGCAT GGCCATCCCAGTCCATCTG (SEQ ID NO: 205)(SEQ ID NO: 206) PNMT  4505921a1 GCAGACCGTAGCCCCAATGGCGTAGTTGTTGCGGAGGTA (SEQ ID NO: 207) (SEQ ID NO: 208)  58488 13477277a1CGGTGCCTCCAAGTGACTG AGGCTGAACTCCTGTGACCTT (SEQ ID NO: 209)(SEQ ID NO: 210) 1tbp4 22759983a1 TATGCTGGTTCCCTGGCTGAGGCCTCATCACACTCGTTG (SEQ ID NO: 211) (SEQ ID NO: 212)  23001 16550629a1TCTTGCGGTGGAACAGAATAAG GCATAGCCCCAAGCAAAAGTT (SEQ ID NO: 213)(SEQ ID NO: 214)   4247  4505163a1 GTGCATAACCGGCCCGAATAAACCGGACAGAAATTCACCCC (SEQ ID NO: 215) (SEQ ID NO: 216) Sdc4  4506861a1GCTCTTCGTAGGCGGAGTC CCTCATCGTCTGGTAGGGCT (SEQ ID NO: 217)(SEQ ID NO: 218) ralb  4506405a1 GCCAACAAGAGTAAGGGCCAGCGTCATACATGAACTGAAGCGTC (SEQ ID NO: 219) (SEQ ID NO: 220) DGKG 4503315a1 GGTGAAGAACGGTGGGTCTC AATCGGCTCATGTGGGTCATA (SEQ ID NO: 221)(SEQ ID NO: 222) TRA2B 21758154a1 CCCCTGCAAAGTCTCGCTCAATCTCGACTGTAAGACCTGCTA (SEQ ID NO: 223) (SEQ ID NO: 224) CLIC114251209a1 ACAACCGCAGGTCGAATTGTT GTGACTCCCTTGAGCCACA (SEQ ID NO: 225)(SEQ ID NO: 226)  90780 23510333a1 CCAGAAAAGAAGCGAAGGAAGTTCCGAAGTCATCTTCAAAAGGG (SEQ ID NO: 227) (SEQ ID NO: 228)   556932483386a1 GCCTTGAAATTAGCAGGTCTTGA CTGTAGAACTTCGTTGTGCATCT(SEQ ID NO: 229) (SEQ ID NO: 230) 151516 22758146a1 TTCGAGAGGCCCCGTTTTCATTGGCCCCATCAAAAGGTTC (SEQ ID NO: 231) (SEQ ID NO: 232) CALD1 13186201a1TTTGAGCGTCGCAGAGAACTT TGTCCCAAGGATTCTTCCTCC (SEQ ID NO: 233)(SEQ ID NO: 234) DUT  2443580a1 CGCCATTTCACCCAGTAAGCAGCCACTCTTCCATAACACCC (SEQ ID NO: 235) (SEQ ID NO: 236) copa 23512328a1TCAGCTTTCACCCCAAAAGAC CACATCCGATAGTCCCATAACTG (SEQ ID NO: 237)(SEQ ID NO: 238) PDCD2 21735594a1 CCGGCCTGCGAGTTTTTAGGGGGGAGGATTCTCAGAAGGT (SEQ ID NO: 239) (SEQ ID NO: 240) 15151622758146a1 TTCGAGAGGCCCCGTTTTC ATTGGCCCCATCAAAAGGTTC (SEQ ID NO: 241)(SEQ ID NO: 242) VGLL4  6633997a1 AATATCGGCATTCTGTGCTACGGCAGGGTCTGTATTCTGGGT (SEQ ID NO: 243) (SEQ ID NO: 244) APOE  4557325a1GTTGCTGGTCACATTCCTGG GGTAATCCCAAAAGCGACCCA (SEQ ID NO: 245)(SEQ ID NO: 246)   8131  6912302a1 CAGCCCCATCAGCGTGATTGCGGCTTACTTGTCTGGGAC (SEQ ID NO: 247) (SEQ ID NO: 248)  10988 21750170a1AGACCCTCCCTCAGTTCCAAT GGGTATTCGCATTCTTGTCCTT (SEQ ID NO: 249)(SEQ ID NO: 250)   9569 15011924a1 CTGCTCTTCAACACACGATACGCCCTCTCTTGACTATCCACGAT (SEQ ID NO: 251) (SEQ ID NO: 252) Acvr11 4557243a1 CCAACCTCCTTCGGAGCAG CTGTGGTGCAGTCACTGTCC (SEQ ID NO: 253)(SEQ ID NO: 254) Adcy4 24497587a1 AGCTGACCTCAGACCCGAGCATACGCCGTGAAGATGACGA (SEQ ID NO: 255) (SEQ ID NO: 256) Calcr1 5031621a1 AAGACCCCATTCAACAAGCAG CCAGTTTCCATCTTGGTCACAG (SEQ ID NO: 257)(SEQ ID NO: 258) Caskin2 24638431a1 CTGATCCTCGCCGTCAAGAATGTTCACGTTGAGCCTCTTTGT (SEQ ID NO: 259) (SEQ ID NO: 260) Ccbp2 13929467a1CTGAGGATGCCGATTCTGAGA TAACGGAGCAAGACCATGAGA (SEQ ID NO: 261)(SEQ ID NO: 262) Cldn5 17939486a1 CTCTGCTGGTTCGCCAACATCAGCTCGTACTTCTGCGACA (SEQ ID NO: 263) (SEQ ID NO: 264) 01839  4503413a1CCCTCCCACTGTATCCACG AGTGACTCTCAAAAGGTCCAGA (SEQ ID NO: 265)(SEQ ID NO: 266) Egf17  7705889a1 CAGCACCTACCGAACCATCTACCCTCCTAGCACTGCATTCAT (SEQ ID NO: 267) (SEQ ID NO: 268) Ehd4 21264315a1CTGCTCTTTGACGCTCACAAG GTCGGCCTTATTCAGCACG (SEQ ID NO: 269)(SEQ ID NO: 270) Entpd1  4741547a1 CAACTATCTGCTGGGCAAATTCAGGCAGGTCTGGATTGAGTTATAC (SEQ ID NO: 271) (SEQ ID NO: 272) Epas116198412a1 TTTCACACGGCACATTTGGAC GTGGACGGGGTCACTATACC (SEQ ID NO: 273)(SEQ ID NO: 274) Erg  4758300a1 CCAGCAGCTCATATCAAGGAAGGTTCCGTAGGCACACTCAAAC (SEQ ID NO: 275) (SEQ ID NO: 276) ESAM1 20452464a1CCCCTGGTGACCAACTTGC TGGGATGAAGACACCTCCCC (SEQ ID NO: 277)(SEQ ID NO: 278) Fgd5 34365081a1 AGCCCCTATGAGTTCTTCCCAGTGCCTGCTCTGATTCTAAACC (SEQ ID NO: 279) (SEQ ID NO: 280) Gpr116 4886491a1 TGCACTGAACTGGAATTACGAG CAGCCGTAGGACTTTTTGTGG (SEQ ID NO: 281)(SEQ ID NO: 282) Hspa12b 31317303a1 CACCCTCGCAGTCTCCAAAAGAAAGCATAGCCACTAGACGTG (SEQ ID NO: 283) (SEQ ID NO: 284) Icam1 4557878a1 TCTGTGTCCCCCTCAAAAGTC GGGGTCTCTATGCCCAACAA (SEQ ID NO: 285)(SEQ ID NO: 286) Icam2  4504557a1 CGGATGAGAAGGTATTCGAGGTCACCCACTTCAGGCTGGTTAC (SEQ ID NO: 287) (SEQ ID NO: 288) Kifc1 33875771a1GAGCCGTGCGAGTTCTCTAC GGCCTTAATCAGAGGTCTCTTCA (SEQ ID NO: 289)(SEQ ID NO: 290) Lats2 18959200a1 ACTTTTCCTGCCACGACTTATTCATCCAGGGAAGTGTCACTGTT (SEQ ID NO: 291) (SEQ ID NO: 292) Lrrk1 14017797a1GCCCGACAACGACATCAAG GCCAAATAGGGTCGAGGAAGTA (SEQ ID NO: 293)(SEQ ID NO: 294) Mmrn2 13376091a1 GGACCCCGTTGGACGTAACCTTGACCTGGTACACTGGCTT (SEQ ID NO: 295) (SEQ ID NO: 296) Myo1b  2764617a1TGGCCTCATTGGAAAGGACC CCAGGCGTTGCTTCCTCAG (SEQ ID NO: 297)(SEQ ID NO: 298) PALD 20521820a1 GGCTGCTGGCAGACTATGGTGGACTTGGCCTTGCTGTTAT (SEQ ID NO: 299) (SEQ ID NO: 300) NM_02351616041779a1 GCTGACCCTGCTTGGCTTAT CCCTCGCCATACCGATGTATTA (SEQ ID NO: 301)(SEQ ID NO: 302) 55332 22450862a1 TGCTTCCTGAGGGGAATGGATCGTGGCTGCACCAAGAAA (SEQ ID NO: 303) (SEQ ID NO: 304) CTTNBP2NL24308179a1 AGCCTGAACTCCTGACACTAT TGCTTTTCGCCATCATTTTTCTC(SEQ ID NO: 305) (SEQ ID NO: 306) CENTD3 21264337a1 GTATGCAGACACGTTCCGACCAGGCGTAGAATGCGTTTCC (SEQ ID NO: 307) (SEQ ID NO: 308) Clorf5413375758a1 ACAGTCACCCCCAGTTATGAT ATCTGGACTAGGTTCCGTTGT (SEQ ID NO: 309)(SEQ ID NO: 310) 134265 18676718a1 TGTGGGTGACAACTGTTCTACCAGAAGCCAATGATACGGGTGAT (SEQ ID NO: 311) (SEQ ID NO: 312) Npr3  4505441a1TGCTCACTTTCTCCCCGTG GGGCAGTAACACCAGCACC (SEQ ID NO: 313)(SEQ ID NO: 314) Pltp 33356541a1 TCACAGAGCTGCAACTGACATAGGCATTGGTGATTTGAAGCA (SEQ ID NO: 315) (SEQ ID NO: 316) Ptprb 30410925a1CATGGTGATTCTTACCTGCTTGA CCCACGACCACTTTCTCATTTT (SEQ ID NO: 317)(SEQ ID NO: 318) Ptprm 18860904a1 TCCAGCAAGAGTAATTCTCCTCCGTACGTGTTGGGTCTCCAGATA (SEQ ID NO: 319) (SEQ ID NO: 320) GRRP119343581a1 TCAAGACGCACCAGGTGATAG CGGTAGAAGATGAGGGAATCAGG(SEQ ID NO: 321) (SEQ ID NO: 322) stard9  7242955a1CTCATGCTTATTCCTCCCATTCC AGGGTGGGTGGATAGTATGTG (SEQ ID NO: 323)(SEQ ID NO: 324) Ramp2  5032021a1 CTGGGCGCTGTCCTGAATCCAATCTCGCAGGGTGCTATAAG (SEQ ID NO: 325) (SEQ ID NO: 326) Rasip127469793a1 TCTGGTGAACGGAAGGAGG CGAAGAAGACTTGACAGAGGC (SEQ ID NO: 327)(SEQ ID NO: 328) Robo4 18676674a1 GTGGGTGAGCAGTTTACTCTGGCCAGGGGTTTCCCATCTTTC (SEQ ID NO: 329) (SEQ ID NO: 330) Sdpr  4759082a1CATCCGGGACAACTCACAGG CTCCAAACTGATCTGTCGCTG (SEQ ID NO: 331)(SEQ ID NO: 332) Slc43a3  6650786a1 TCAGCCCCGAGGATGGTTTAAGGCTAAGTGCAAGGAGACA (SEQ ID NO: 333) (SEQ ID NO: 334) Slc9a3r2 2047328a1 GCTCCGAAGCTGGCAAGAA GGGACTTGTCACTATGCAGGTT (SEQ ID NO: 335)(SEQ ID NO: 336) Slco2a1 26996627a1 GGGCAGCGACACCTCTACTATGGAAATGAGACCCGATGAAGAA (SEQ ID NO: 337) (SEQ ID NO: 338) B2M  4757826a1GGCTATCCAGCGTACTCCAAA CGGCAGGCATACTCATCTTTTT (SEQ ID NO: 339)(SEQ ID NO: 340) HPRT1  4504483a1 CCTGGCGTCGTGATTAGTGATAGACGTTCAGTCCTGTCCATAA (SEQ ID NO: 341) (SEQ ID NO: 342) RPL13A 6912634a1 CGAGGTTGGCTGGAAGTACC CTTCTCGGCCTGTTTCCGTAG (SEQ ID NO: 343)(SEQ ID NO: 344) GAPDH  7669492a1 ATGGGGAAGGTGAAGGTCGGGGGTCATTGATGGCAACAATA (SEQ ID NO: 345) (SEQ ID NO: 346) ACTB  4501885a1CATGTACGTTGCTATCCAGGC CTCCTTAATGTCACGCACGAT (SEQ ID NO: 347)(SEQ ID NO: 348)

TABLE 14 EFFECTS OF ANTIANGIOGENIC SMs ON ANGIOGENEIS GENE EXPRESSIONNSC: 19630 122567 150117 259969 292222 292596 AKT1  0.1 −1.6  1.6 −1.4 0.7 −0.8  ANGPT1 −0.5  1.6  1.7 1.4 0.9 2.2 ANGPT2 −1.8 −0.9 −1.2 0.9−1.3  0.8 ANGPTL3 −5.0 −1.7  1.8 3.6 0.6 1.0 ANGPTL4 −0.2 −0.4 −4.0 1.51.3 −3.2  ANPEP −0.4 −1.2 −1.7 0.2 −0.6  0.8 BAL1 −0.9  0.2  0.8 0.4 0.21.7 CCL11 −0.3  0.6  0.7 0.0 0.2 2.1 CCL2  2.3  2.5  0.9 3.6 0.9 2.4CDH5 −0.2 −1.9 −1.6 −1.6  −0.1  −0.2  COL18A1  0.4  1.6  0.8 1.9 1.2 2.3COL4A3  0.1  1.9  2.4 3.2 2.0 1.9 DLL4 −4.5 −8.7  5.5 −4.1  −2.2  −6.6 CXCL10  4.7  3.7 −1.7 2.8 0.8 2.5 CXCL3 12.1  4.0  0.8 11.6  5.0 −3.8 CXCL5  4.8  4.3 −3.5 10.2  3.4 5.6 CXCL6 −0.5  1.1  1.2 1.0 0.4 2.2CXCL9 −1.0  1.6  1.2 0.5 0.4 1.5 TYMP  0.3 −0.3 −1.1 0.1 −0.1  −5.5 S1PR1 −2.6 −0.7 −1.9 −0.3  −0.3  0.2 EFNA1  3.7 −0.3  0.4 7.8 0.9 3.1EFNA3 −0.4 −0.4 −1.8 0.3 0.1 0.5 EFNB2 −1.9 −4.0  0.4 −6.6  −2.2  −3.4 EGF −1.0 −0.3 −2.3 0.2 2.8 2.2 ENG  0.1 −1.7 −0.1 −0.7  −0.9  −0.3 EPHB4 −0.8 −0.5 −1.2 0.3 −0.5  0.9 EREG  7.6  1.9 −2.4 9.3 5.0 9.6 FGF1−3.0  1.9 −3.8 5.7 −1.4  4.8 FGF2  1.7 −0.7  0.0 2.5 −0.6  2.5 FGFR3−0.2  1.2  1.0 1.2 0.8 2.2 FIGF −3.2 −0.1  2.4 0.2 0.1 1.7 FLT1 −2.1−4.4 −0.5 −0.7  −0.2  −1.9  HAND2 −0.5  0.4  1.5 1.4 1.0 1.7 HGF −0.3 1.3  1.5 1.5 1.3 2.2 HIF1A −1.7 −3.1 −1.8 0.7 −0.7  −2.1  HPSE  0.6−8.3 −0.2 2.6 1.6 −0.3  ID1  1.0 −0.2  0.3 1.5 0.0 4.0 ID3  0.4 −0.3 0.5 2.5 −0.2  1.0 IFNA1  0.4  1.1  1.9 1.1 0.8 2.2 IFNB1 −0.4  1.2  2.11.6 1.1 2.6 IFNG  4.7  8.3 −1.9 6.0 3.1 6.5 IGF1  3.6 −0.1 −0.2 −2.6 6.0 0.6 IL1B  0.2 −0.1  9.5 1.3 4.3 −4.1  B2M  0.0  0.1  2.8 1.2 −0.4 −2.0  HPRT1  0.8  0.0 −0.4 0.6 0.3 −0.5  RPL13A −0.2  0.7 −0.8 1.2 0.71.9 GADPH  0.1 −0.7 −1.3 −1.0  −0.1  0.3 ACTB  0.1 −1.0 −0.9 −2.5  −0.8 0.0 IL6  3.5 14.4  2.2 9.4 3.0 3.7 IL8  3.0  7.4  6.0 11.3  13.9  5.1ITGAV −0.3 −2.7 −1.8 −0.9  1.2 −0.9  ITGB3 −0.9 −1.4 −1.4 −2.9  2.5−1.8  JAG1 −1.2  0.6  0.3 1.7 1.6 0.5 KDR −2.7 −2.3 −2.1 −0.5  −1.2 −1.8  LAMAS5 −0.8 −1.1 −2.2 −0.8  −0.4  −0.3  LECT1 −0.4  0.3 −0.2 −8.0 1.4 1.4 LEP −4.5  0.2 −0.2 0.8 −0.6  2.6 MDK −1.1 −0.9 −1.5 −1.0  0.8−1.5  MMP2  0.5  0.3 −0.6 −0.1  2.8 1.1 MMP9  0.3  0.9 −0.3 4.3 3.3 1.8Notch4 −1.8 −1.1 −0.7 −0.3  −0.4  −0.6  NRP1 −0.5 −3.0 −2.0 −2.6  0.8−2.3  NRP2 −2.2 −4.3 −3.2 −3.5  1.0 −3.2  PDGFA −0.7 −1.7 −0.9 1.1 1.64.0 PECAM1 −2.6 −2.7 −2.5 −3.0  −0.7  −1.8  PGF −2.4 −4.9 −1.6 −7.2  0.6−1.7  PLAU  0.1 −0.1  0.0 3.8 2.7 −0.2  PLG −2.7 −1.5 −3.9 −1.0  −0.7 −0.4  PLXDC1 −4.0  0.7 −3.2 2.3 −0.1  0.3 PROK2 −1.3  1.3  0.7 2.2 1.92.0 PTGS1 −2.4  0.8  0.3 2.9 2.0 1.1 SERPINF1 −0.8  0.2  1.5 6.5 2.3 0.7SPHK1 −0.5  0.9  1.2 3.2 2.0 1.8 STAB1 −1.0 −1.7 −0.9 0.5 0.2 −0.5  TEK−0.4  0.3  0.7 1.6 2.0 1.3 TGFA  1.1  2.0  5.3 8.7 −3.4  8.9 TGFB1 −1.3−1.2 −1.3 0.3 1.1 −0.8  TGFB2 −0.5  1.2  1.6 1.3 1.9 2.1 TGFBR1 −0.1 1.2  1.2 1.1 1.8 1.4 THBS1  0.5  0.1 −0.6 −0.1  1.1 −0.1  THBS2 −0.6 1.2  0.9 2.6 1.9 1.1 TIMP1 −0.8 −1.4 −0.7 2.5 1.6 1.4 TIMP2 −0.9 −1.4−0.8 0.0 1.1 0.4 TIMP3  0.7 −4.3 −2.7 −0.6  −0.4  −1.2  TNF −0.6 −0.2 2.4 9.3 1.0 6.1 TNFAIP2  0.2 −1.0 −0.6 4.6 0.6 −0.2  TNFAIP2 −0.4 −1.1 2.7 4.7 0.2 −0.1  VEGFA  4.2  4.9 −0.3 5.7 3.2 0.4 VEGFC  0.1  1.8  2.01.9 2.7 1.6 ROBO4  0.1  0.0 −0.2 −4.1  1.1 −1.2  EphB1  0.0  0.8  0.70.6 0.9 0.4 B2M −0.1 −0.2 −1.2 1.5 1.3 −1.6  HPRT1  0.9  0.0  0.6 −0.5 1.4 0.1 RPL13A  0.3  1.2  0.1 0.7 1.7 1.5 GADPH −0.3 −0.8 −0.1 0.0 1.40.8 ACTB −0.3 −1.0 −0.6 −2.2  −6.3  −0.4  CD248  1.3  1.2 −0.4 0.6 1.30.2 GPCR124 −1.7 −3.7 −2.9 −4.6  −1.9  −6.7  PLXDC1 −5.7 −1.7 −0.6 −2.4 0.3 0.2 ANTXR1 −0.3 −3.5 −4.2 −1.4  −0.2  −2.1  RASD2  0.9  0.5 −0.1 0.50.3 −0.5  ARHGEF17  0.2 −5.0 −2.0 −0.8  −0.9  −1.6  TNS3 −1.8  2.0  0.6−8.0  0.5 1.3 DKK3 −1.5 −1.9 −3.3 −2.7  −1.1  −0.4  MMP11  0.8  1.3 −0.20.1 0.1 −0.3  NID1 −2.6 −6.3 −5.2 −3.6  −2.5  −3.6  THY1 −11.3  −9.4−12.7  −3.0  −8.0  −5.8  CST4  0.8  1.3 −0.1 0.4 0.8 0.3 MRC2  1.6  0.7−0.7 −0.6  0.6 −0.5  TNS1  0.6  0.0 −0.9 −0.3  −0.3  −0.5  BMP1  0.5 0.9 −0.4 −0.6  0.4 −0.4  COMT −0.7 −1.5 −1.0 −1.7  −0.2  −0.7  PTPRCAP 0.4  0.0  0.2 0.9 0.1 −0.5  57722  0.2  0.1 −0.9 −0.1  0.0 −0.2  EXTL3 1.0  0.3  0.0 0.4 0.8 −0.5  Vwf −1.8 −3.5 −3.7 −5.4  −3.3  −2.6  PNMT 0.7  1.1 −0.4 −0.1  0.1 −0.7  58488  1.2  1.5  0.3 0.4 1.0 0.2 itbp4−0.8 −1.9 −2.5 −1.7  0.1 −1.5  23001  0.4 −0.5 −1.0 0.3 −0.8  0.1 4247 1.0  0.3 −0.1 0.2 0.5 −0.3  Sdc4  2.2  0.4  0.2 4.0 0.7 1.4 ralb  0.1 0.3 −0.7 0.7 −0.4  −0.5  DGKG  5.4  2.0  3.4 6.1 2.9 1.8 TRA2B  1.5−1.6 −0.2 0.6 −0.6  0.9 CLIC1 −0.1  0.9  0.0 1.8 6.6 0.6 90780 −1.2 −1.9−1.4 −2.4  0.1 −0.8  5569 −3.0  0.8 −0.1 4.6 1.5 0.1 151516  1.2  1.1 0.0 0.5 0.7 0.3 CALD1  1.7  2.3  1.1 −0.6  0.6 1.6 DUT −0.8 −1.8 −0.2−1.9  −0.4  −1.2  copa  0.4  0.1  0.0 −2.1  0.2 −0.5  PDCD2  0.5 −0.4−0.9 −1.7  0.2 −1.1  151516  0.9  1.3  0.0 0.5 0.7 0.3 VGLL4 −1.3 −3.5−3.5 1.3 −0.8  −2.8  APOE  3.3  0.8 −1.0 0.8 −2.2  5.1 8131  0.6  0.1−0.5 −0.2  0.1 0.3 10988  2.5  1.2  0.8 0.3 1.0 0.9 9569 −2.3 −2.2 −4.2−1.7  0.2 −2.0  B2M −1.4 −0.2 −0.7 0.5 0.0 −0.4  HPRT1  0.2 −0.6  1.10.8 0.2 0.4 RPL13A  1.0  0.9  0.6 0.0 0.3 1.1 GADPH  0.1 −0.7 −0.5 −1.7 −0.1  −0.3  ACTB −0.9 −0.5 −0.5 −2.2  −0.9  −0.9  Acvrl1 −0.6 −1.3 −1.2−1.9  0.0 −1.6  Adcy4  0.0 −0.4 −0.5 −0.6  −0.4  4.3 Calcrl −4.3 −4.0−5.4 −2.9  −1.1  −4.3  Caskin2 −2.8 −2.3 −2.7 −2.3  −6.7  −2.3  Ccbp2−0.1  1.4  0.4 5.6 0.4 0.0 Cldn5  0.6  0.0  0.1 0.4 −0.1  −0.6  1839−1.2  1.0  0.9 −4.2  −0.1  −0.3  Egfl7 −3.8 −2.6 −3.3 −2.3  −1.2  −3.3 Ehd4 −1.1 −0.8 −0.6 −0.7  −1.4  −1.0  Entpd1 −4.9 −0.3 −8.9 −5.6  −5.5 −8.3  Epas1  1.8  0.9  0.4 1.5 −1.7  0.3 Erg −1.6  2.4 −3.2 −3.9  3.7−6.4  ESAM1 −3.3 −2.9 −2.6 −1.6  −1.1  −2.0  Fgd5 −1.6 −1.5 −1.2 −0.8 −0.8  −1.0  Grp116 −1.9 −2.3 −3.1 −5.5  −4.4  −3.1  Hspa12b −4.6 −3.4−3.5 −10.2  −2.2  −3.3  Icam1  0.1  0.0  0.6 2.8 0.5 0.5 Icam2  0.4  0.1 0.0 7.5 0.1 −0.4  Kifc1  3.4  1.1 −0.1 −4.8  0.5 1.6 Lasts2 −0.1  4.8 0.3 1.5 0.1 0.4 Lrrk1 −2.9 −0.5 −1.8 0.3 −0.1  −1.5  Mmm2 −2.4 −3.5−3.4 −7.5  −1.0  −2.4  Myo1b −2.3 −2.9 −2.7 −3.0  −1.5  −2.0  PALD −1.1−0.5 −1.7 −1.3  1.8 −1.9  NM_023516  0.5  0.2  0.6 0.9 0.4 0.2 55332−1.1 −1.0 −1.7 0.1 2.4 −8.6  CTTNBP2NL −2.3 −0.9 −2.3 −1.0  0.2 −3.4 CENTD3  0.6  1.1 −0.2 −1.0  −0.5  0.1 C1orf54 −4.8 −3.4 −1.3 −6.1  −0.1 −2.2  134265 −1.6 −0.2 −1.0 −0.6  −1.3  −1.6  Npr3  8.3  1.5  0.5 2.70.9 0.7 Pltp  0.9  1.5  0.9 1.4 0.4 0.5 Ptprb −1.6  0.5 −2.6 −0.1  0.3−1.9  Ptprm −1.2 −0.4 −1.4 1.7 −0.1  −0.8  GRRP1  0.0 −0.3  0.0 0.6 0.10.2 stard9 −0.7  0.5  0.1 0.0 0.8 −0.9  RAMP2 −4.8 −5.8 −4.4 −5.9  −0.6 −5.0  Rasip1  3.1 −1.2 −1.7 −1.1  −0.3  −0.5  Robo4  0.3  1.2 −0.3 0.30.0 0.2 Sdpr −0.1  1.6  0.0 0.9 −4.9  0.8 Slc43a3 −0.8  1.8  0.0 0.2 1.00.6 Slc9a3r2 −0.5 −1.9 −2.6 −0.8  −2.4  −4.4  Slco2a1 −2.5 −0.6 −2.9−2.4  −5.3  −1.0  B2M −0.9  0.3 −0.8 0.8 0.6 −0.8  HPRT1  2.0 −0.2  0.40.5 0.4 0.1 RPL13A  0.4  1.3  0.2 0.0 1.2 0.6 GADPH −1.6 −2.0 −0.5 −2.1 −0.2  0.4 ACTB −1.6 −0.1 −0.2 0.4 −0.6  −0.1  Log2 fold-change of geneexpression. Fold-changes with P ≦ 0.001 are underlined.Clustering Analysis of Gene Expression Data

It is well known that tumors become resistant to antiangiogenic therapy(Bergers and Hanahan, Nature Reviews: Cancer, 8:592-603, 2008). Tumorangiogenesis involves multiple pathways. Current antiangiogenic drugs(such as AVASTIN®) successfully inhibit one pathway and are followed byactivation of alternative pathways which resume the angiogenesis processwithin the tumor. A multitargeted strategy that inhibits multipleangiogenesis pathways is expected to more successfully avoid drugresistance.

To that end, clustering analysis was performed with the gene expressiondata to identify drugs which inhibit angiogenesis, and by extensiontumor growth, by targeting different sets of genes and thereforedifferent angiogenesis pathways.

Combinations of drugs which effect distant gene sets in the clusteringanalysis could potentially target different angiogenesis pathways andtherefore be more efficient antiangiogenic regimens. Several potentialdrug combinations emerge from this clustering analysis, such as NSC259969+NSC 150117.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as a limitation onthe scope of the invention. Rather, the scope of the invention isdefined by the following claims. We therefore claim as our invention allthat comes within the scope and spirit of these claims.

The invention claimed is:
 1. A method of inhibiting endothelial celltube formation in a subject, comprising administering to the subject aneffective amount of a composition comprising2-benzylidene-3-(cyclohexylamino)-3H-inden-1-one chloride (NSC 150117),or a pharmaceutically acceptable salt thereof, thereby inhibitingendothelial cell tube formation in the subject.
 2. The method of claim1, wherein the composition further comprises[4-[(4-arsonophenyl)methyl]phenyl]arsonic acid (NSC 48300), or apharmaceutically-acceptable salt thereof.
 3. The method of claim 1,wherein the composition is administered topically, intravenously,orally, parenterally, or as an implant.
 4. The method of claim 1,further comprising administering to the subject an inhibitor of bFGF,FGF, or VEGF.